REAGENTS AND METHODS FOR DETECTING AAV SHEDDING
20220349017 · 2022-11-03
Assignee
Inventors
- Arejas J. Uzgiris (Berkeley, CA)
- Isabelle Philipp (Berkeley, CA, US)
- Xiaolei Qiu (San Ramon, CA, US)
- Sara Weitz (El Sobrante, CA, US)
- Zuguang Wang (Hayward, CA, US)
Cpc classification
C12Q1/6818
CHEMISTRY; METALLURGY
International classification
Abstract
The present disclosure provides, among other things, primers and probes for detecting shedding of an AAV construct or fragment thereof in a subject. In some embodiments the primers are selected to generate an amplicon that comprises (i) a first strand comprising (1) a nucleotide sequence corresponding to the forward primer, and (2) a nucleotide sequence corresponding to a portion of the AAV construct or fragment thereof, (ii) a second strand comprising (1) a nucleotide sequence of the reverse primer, and (2) a nucleotide sequence that is complementary to the portion of the AAV construct or fragment thereof, or (iii) a combination thereof, where the portion of the AAV construct or fragment thereof comprises a nucleotide sequence that spans a junction between a regulatory element and the therapeutic gene of interest.
Claims
1. A composition comprising a collection of primers that comprises: (a) one or more forward primers comprising a forward primer with a nucleotide sequence according to SEQ ID NO: 1 or an active fragment thereof; and (b) one or more reverse primers comprising a reverse primer with a nucleotide sequence according to SEQ ID NO: 11 or an active fragment thereof.
2. The composition of claim 1, further comprising: one or more probes comprising a probe with a nucleotide sequence according to SEQ ID NO: 17 or an active fragment thereof.
3. The composition of claim 1, wherein the one or more forward primers, the one or more reverse primers, the one or more probes or a combination thereof is DNA.
4. The composition of claim 1, wherein the one or more forward primers, the one or more reverse primers, the one or more probes, or a combination thereof comprises a detectable label.
5. The composition of claim 4, wherein the detectable label does not comprise nucleotides.
6. The composition of claim 4, wherein the detectable label is a fluorescent moiety.
7. The composition of claim 6, wherein the probe comprises a fluorescent moiety.
8. The composition of claim 7, wherein the probe comprises one or more quenchers.
9. The composition of claim 1, wherein an active fragment is (a) at least 10 nucleotides in length; (b) at least 15 nucleotides in length; or (c) at least 20 nucleotides in length.
10. The composition of claim 9, wherein the composition further comprises a sample obtained from a subject who has been administered a Duchenne Muscular Dystrophy (DMD) AAV construct, wherein the DMD AAV construct comprises one or more regulatory elements and a therapeutic gene of interest, and wherein the sample comprises nucleic acids.
11. The composition of claim 10, wherein the nucleic acids in the sample comprise the DMD AAV construct or a fragment thereof.
12. The composition of claim 10, wherein the one or more regulatory elements comprises an enhancer, a promoter, a polyA signal sequence, or a combination thereof.
13. The composition of claim 10, wherein the therapeutic gene of interest comprises microdystrophin.
14. The composition of claim 10, further comprising a plurality of amplicons, wherein each amplicon comprises: (a) a first strand comprising: (i) a nucleotide sequence corresponding to the forward primer, and (ii) a nucleotide sequence corresponding to a portion of the DMD AAV construct or fragment thereof; (b) a second strand comprising: (i) a nucleotide sequence of the reverse primer, and (ii) a nucleotide sequence that is complementary to the portion of the DMD AAV construct or fragment thereof; or (c) a combination thereof; and wherein the portion of the DMD AAV construct or fragment thereof comprises a nucleotide sequence that spans a junction between two regulatory elements or a junction between a regulatory element and the therapeutic gene of interest.
15. The composition of claim 14, wherein the probe with a nucleotide sequence according to SEQ ID NO: 17 or an active fragment thereof is capable of hybridizing with the amplicon.
16. The composition of claim 15, wherein a level of probe-amplicon hybridization is detectable.
17. The composition of claim 15, wherein a level of probe-amplicon hybridization is quantifiable.
18. A method comprising: (a) contacting a sample obtained from a subject with a composition according to claim 1, wherein the subject has been administered a DMD AAV construct, wherein the DMD AAV construct comprises one or more regulatory elements and a therapeutic gene of interest; and (b) amplifying a target sequence to generate a plurality of amplicons, wherein the target sequence is a DMD AAV construct or fragment thereof and each amplicon comprises: (i) a first strand comprising: (1) a nucleotide sequence corresponding to the forward primer, and (2) a nucleotide sequence corresponding to a portion of the DMD AAV construct or fragment thereof; (ii) a second strand comprising: (1) a nucleotide sequence of the reverse primer, and (2) a nucleotide sequence that is complementary to the portion of the DMD AAV construct or fragment thereof; or (iii) a combination thereof; and wherein the portion of the DMD AAV construct or fragment thereof comprises a nucleotide sequence that spans a junction between two regulatory elements or a junction between a regulatory element and the therapeutic gene of interest.
19. The method of claim 18, wherein the composition comprises a plurality of probes each with a nucleotide sequence according to SEQ ID NO: 17 or an active fragment thereof.
20. The method of claim 19, further comprises detecting a level of hybridization between the plurality of probes and the plurality of amplicons.
21. The method of claim 20, wherein the level of hybridization between the plurality of probes and the plurality of amplicons indicates a quantity of DMD AAV construct in the sample.
22. A composition comprising a collection of primers that comprises: (a) one or more forward primers comprising a forward primer with a nucleotide sequence according to SEQ ID NO: 35 or an active fragment thereof; and (b) one or more reverse primers comprising a reverse primer with a nucleotide sequence according to SEQ ID NO: 45 or an active fragment thereof.
23. The composition of claim 22, further comprising: one or more probes comprising a probe with a nucleotide sequence according to SEQ ID NO: 55 or an active fragment thereof.
24. The composition of claim 22, wherein the one or more forward primers, the one or more reverse primers, the one or more probes, or a combination thereof is DNA.
25. The composition of claim 22, wherein the one or more forward primers, the one or more reverse primers, the one or more probes, or a combination thereof comprises a detectable label.
26. The composition of claim 25, wherein the detectable label does not comprise nucleotides.
27. The composition of claim 25, wherein the detectable label is a fluorescent moiety.
28. The composition of claim 22, wherein the probe comprises a fluorescent moiety.
29. The composition of claim 28, wherein the probe comprises one or more quenchers.
30. The composition of claim 22, wherein an active fragment is (a) at least 10 nucleotides in length; (b) at least 15 nucleotides in length; or (c) at least 20 nucleotides in length.
31. The composition of claim 22, wherein the composition further comprises a sample obtained from a subject has been administered a hemophilia B (Hem-B) AAV construct, wherein the Hem-B AAV construct comprises one or more regulatory elements and a therapeutic gene of interest, and wherein the sample comprises nucleic acids.
32. The composition of claim 31, wherein the nucleic acids in the sample comprise a Hem-B AAV construct or a fragment thereof.
33. The composition of claim 31, wherein the one or more regulatory elements comprises an enhancer, a promoter, a polyA signal sequence, or a combination thereof.
34. The composition of claim 31, wherein the therapeutic gene of interest comprises Factor IX.
35. The composition of claim 31, wherein the composition further comprises a plurality of amplicons, wherein each amplicon comprises: (a) a first strand comprising: (i) a nucleotide sequence corresponding to the forward primer, and (ii) a nucleotide sequence corresponding to a portion of the Hem-B AAV construct or fragment thereof; (b) a second strand comprising: (i) a nucleotide sequence of the reverse primer, and (ii) a nucleotide sequence that is complementary to the portion of the Hem-B AAV construct or fragment thereof; or (c) a combination thereof; and wherein the portion of the Hem-B AAV construct or fragment thereof comprises a nucleotide sequence that spans a junction between two regulatory elements or a junction between a regulatory element and the therapeutic gene of interest.
36. The composition of claim 35, wherein the probe with a nucleotide sequence according to SEQ ID NO: 55 or an active fragment thereof is capable of hybridizing with the amplicon.
37. The composition of claim 36, wherein a level of probe-amplicon hybridization is detectable.
38. The composition of claim 36, wherein a level of probe-amplicon hybridization is quantifiable.
39. A method comprising: (a) contacting a sample obtained from a subject with a composition according to claim 22, wherein the subject has been administered a Hem-B AAV construct, wherein the Hem-B AAV construct comprises one or more regulatory elements and a therapeutic gene of interest; and (b) amplifying a target sequence to generate a plurality of amplicons, wherein the target sequence is a Hem-B AAV construct or fragment thereof and each amplicon comprises: (i) a first strand comprising: (1) a nucleotide sequence corresponding to the forward primer, and (2) a nucleotide sequence corresponding to a portion of a strand of the Hem-B AAV construct or fragment thereof; (ii) a second strand comprising: (1) a nucleotide sequence of the reverse primer, and (2) a nucleotide sequence that is complementary to a portion of a strand of the Hem-B AAV construct or fragment thereof; or (iii) a combination thereof; and wherein the portion of the Hem-B AAV construct or fragment thereof comprises a nucleotide sequence that spans a junction between two regulatory elements or a junction between a regulatory element and the therapeutic gene of interest.
40. The method of claim 39, wherein the composition comprises a plurality of probes each with a nucleotide sequence according to SEQ ID NO: 55 or an active fragment thereof.
41. The method of claim 40, further comprises detecting a level of hybridization between the plurality of probes and the plurality of amplicons.
42. The method of claim 41, wherein the level of hybridization between the plurality of probes and the plurality of amplicons indicates a quantity of DMD AAV construct in the sample.
43. A method comprising: (a) contacting a sample obtained from a subject with a composition comprising a first primer and a second primer, wherein the subject has been previously administered an AAV construct comprising one or more regulatory elements and a gene of interest, wherein the first primer comprises a sequence corresponding to or complementary to the gene of interest and the second primer comprises a sequence corresponding to or complementary to a regulatory element; and (b) performing a polymerase chain reaction to generate a plurality of amplicons, wherein the target sequence is the AAV construct or fragment thereof and each amplicon comprises: (i) a first strand comprising: (1) a nucleotide sequence corresponding to the forward primer, and (2) a nucleotide sequence corresponding to a portion of the AAV construct or fragment thereof; (ii) a second strand comprising: (1) a nucleotide sequence of the reverse primer, and (2) a nucleotide sequence that is complementary to the portion of the AAV construct or fragment thereof; or (iii) a combination thereof; wherein the portion of the AAV construct or fragment thereof comprises a nucleotide sequence that spans a junction between a regulatory element and the therapeutic gene of interest.
44. A method comprising: (a) contacting a sample obtained from a subject with a composition comprising a first primer and a second primer, wherein the subject has been previously administered an AAV construct comprising two or more regulatory elements and a gene of interest, wherein the first primer comprises a sequence corresponding to or complementary to a first regulatory element and the second primer comprises a sequence corresponding to or complementary to a second regulatory element; and (b) performing a polymerase chain reaction to generate a plurality of amplicons, wherein the target sequence is the AAV construct or fragment thereof and each amplicon comprises: (i) a first strand comprising: (1) a nucleotide sequence corresponding to the forward primer, and (2) a nucleotide sequence corresponding to a portion of the AAV construct or fragment thereof; (ii) a second strand comprising: (1) a nucleotide sequence of the reverse primer, and (2) a nucleotide sequence that is complementary to the portion of the AAV construct or fragment thereof; or (iii) a combination thereof; wherein the portion of the AAV construct or fragment thereof comprises a nucleotide sequence that spans a junction between the first regulatory element and the second regulatory element.
Description
BRIEF DESCRIPTION OF THE DRAWING
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DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
Gene Therapy
[0115] Gene therapy refers to techniques that involve the introduction of gene(s) or modification of gene expression to treat, ameliorate, and/or prevent disease. There are several approaches to gene therapy, including but not limited to: replacing a mutated gene that causes disease with a healthy copy of the gene; inactivating, or “knocking out,” a mutated gene that is functioning improperly; and/or introducing a gene into the body to help fight a disease. Gene therapy is a promising treatment option for a number of diseases (including inherited disorders, some types of cancer, and certain viral infections). Generally, gene therapy is tested for diseases that have a known genetic association and/or for diseases where the current standard of care is considered lacking.
[0116] Clinical trials involving gene therapy techniques are subject to regulations, which can require certain monitoring guidelines are met. One such regulation involving viral delivery methods requires an accurate and often continual measuring of viral dosage and viral “shedding” (the viral particles released through any mechanism from the test subject). The present disclosure provides compositions and methods that aid in the essential monitoring of viral shedding, providing physicians and scientists with robust and reliable measurements.
Exemplary Conditions
[0117] Duchene Muscular Dystrophy
[0118] Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder that affects approximately 1 in 5000 live male births (Mendell et al., 2012; and Moat et al., 2013; both of which are incorporated herein by reference for any purpose). It was first described in detail in the 1860s by the French neurologist Guillaume-Benjamin-Amand Duchenne (Duchenne 1861, which is incorporated herein by reference for any purpose). Patients with Duchenne muscular dystrophy usually exhibit motor symptoms within the first 3 years of life. Most commonly, they may have a “waddling” gait that results from hip-girdle weakness and require the use of their hands when they get up from the floor (Gower's maneuver).
[0119] The disease is due to an absence of the dystrophin protein in the skeletal muscle membrane, and muscles lacking dystrophin are more susceptible to mechanical injury. Absence of dystrophin may be demonstrated by the absence of immunostaining for dystrophin on muscle biopsy. Genetic testing, however, has become more readily available in recent years and has become the standard method of diagnostic confirmation. Typically, genetic testing starts with screening for duplications or deletions either by multiplex ligation-dependent probe amplification (MLPA) or by microarray analysis. If duplication/deletion testing is negative, then sequencing of all 79 exons is performed to detect missense, nonsense, splice site, and small indel mutations (Birnkrant et al., part 3, 2018; which is incorporated herein by reference for any purpose). Dependent upon the genetic testing results, various gene therapy avenues may be available.
[0120] The DMD gene is considered one of the largest genes in the human genome, and many disruptive mutations have been reported. De novo mutations appear to be common, with estimates ranging between 12 and 33% of patients with DMD (Shieh, 2018; which is incorporated herein by reference for any purpose). Estimates of the prevalence of different mutation types vary, but reports suggest that, among DMD patients, 69% have large deletions, 11% have large duplications, 10% have nonsense mutations, 7% have missense or small indels, and another 3% have intronic or other mutations (Shieh, 2018; which is incorporated herein by reference for any purpose). The large size of the dystrophin protein means that full gene replacement therapy for DMD is only feasible with large viruses such as adenoviral vectors. Alternatively, rather than introducing the entirety of the large DMD gene, miniaturized but efficacious versions of dystrophin, often nicknamed “minidystrophins” or “microdystrophins”, can be employed. These mini-genes are often small enough to be packaged in an adeno-associated virus (AAV). Over the past 16 years, the preclinical and clinical investigational experience with AAVs has grown to provide a better understanding of the safety profile of these viruses. This experience and knowledge base has made AAV vector delivery of gene therapies a key clinical candidate, including for the treatment of muscular dystrophy through the delivery of micro/minidystrophin genes. While AAV based gene therapy may be a breakthrough, there remains a pressing need for accurate and reproducible materials and methods for monitoring AAV shedding in patients being treated for DMD using gene therapy.
[0121] Hemophilia B
[0122] Hemophilia B, also called factor IX (FIX) deficiency or Christmas disease, is a genetic disorder caused by missing or defective Factor IX, a clotting protein. Although it is passed down from parents to children, about ⅓ of cases are caused by de-novo mutations. According to the US Centers for Disease Control and Prevention, hemophilia occurs in approximately 1 in 5,000 live births. There are about 20,000 people with hemophilia in the US. All races and ethnic groups are affected. Hemophilia B is four times less common than hemophilia A. People with hemophilia B bleed longer than other people. Bleeds can occur internally, into joints and muscles, or externally, from minor cuts, dental procedures or trauma. How frequently a person bleeds and how serious the bleeds are depends on how much FIX is in the plasma, Hemophilia B is tested for using assays that evaluate clotting time. Additionally, genetic testing is utilized to determine the underlying molecular mechanism of an individual's hemophilia B, as there are over 1100 unique mutations known to cause hemophilia B.
[0123] The main medication to treat hemophilia B is concentrated FIX product, called clotting factor or simply factor. Recombinant factor products, which are developed in a lab through the use of DNA technology preclude the use of human-derived pools of donor-sourced plasma. While plasma-derived FIX products are still available, approximately 75% of the hemophilia community takes a recombinant FIX product. These factor therapies are infused intravenously through a vein in the arm or a port in the chest, and often patients require routine costly, time consuming, and burdensome treatment. Gene therapy is proving to be amenable and potentially highly efficacious for the treatment of hemophilia B. However while AAV based gene therapy may be a breakthrough, there remains a pressing need for accurate and reproducible materials and methods for monitoring AAV shedding in patients being treated for hemophilia B using gene therapy.
Viral Vectors for Therapeutic Uses
[0124] Among other things, the present disclosure provides compositions and methods for measuring polynucleotides. Polynucleotide constructs according to the present disclosure include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viral constructs (e.g., lentiviral, retroviral, adenoviral, and adeno-associated viral constructs) that incorporate a polynucleotide sequence of interest or characteristic portion thereof.
[0125] Those of skill in the art will be capable of selecting suitable constructs for measurement. In some embodiments, a construct is a viral construct. In some embodiments, a viral construct is a lentivirus, retrovirus, adenovirus, or adeno-associated virus construct. In some embodiments, a construct is an adeno-associated virus (AAV) construct (see, e.g., Asokan et al., Mol. Ther. 20: 699-7080, 2012, which is incorporated in its entirety herein by reference). In some embodiments, a viral construct is an adenovirus construct. In some embodiments, a viral construct may also be based on or derived from an alphavirus. Alphaviruses include Sindbis (and VEEV) virus, Aura virus, Babanki virus, Barmah Forest virus, Bebaru virus, Cabassou virus, Chikungunya virus, Eastern equine encephalitis virus, Everglades virus, Fort Morgan virus, Getah virus, Highlands J virus, Kyzylagach virus, Mayaro virus, Me Tri virus, Middelburg virus, Mosso das Pedras virus, Mucambo virus, Ndumu virus, O'nyong-nyong virus, Pixuna virus, Rio Negro virus, Ross River virus, Salmon pancreas disease virus, Semliki Forest virus, Southern elephant seal virus, Tonate virus, Trocara virus, Una virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, and Whataroa virus. Generally, the genome of such viruses encode nonstructural (e.g., replicon) and structural proteins (e.g., capsid and envelope) that can be translated in the cytoplasm of the host cell. Ross River virus, Sindbis virus, Semliki Forest virus (SFV), and Venezuelan equine encephalitis virus (VEEV) have all been used to develop viral constructs for coding sequence delivery. Pseudotyped viruses may be formed by combining alphaviral envelope glycoproteins and retroviral capsids. Examples of alphaviral constructs can be found in U.S. Publication Nos. 20150050243, 20090305344, and 20060177819; constructs and methods of their making are incorporated herein by reference to each of the publications in its entirety.
[0126] Constructs suitable for measurement may be of different sizes. In some embodiments, a construct is a plasmid and can include a total length of up to about 1 kb, up to about 2 kb, up to about 3 kb, up to about 4 kb, up to about 5 kb, up to about 6 kb, up to about 7 kb, up to about 8 kb, up to about 9 kb, up to about 10 kb, up to about 11 kb, up to about 12 kb, up to about 13 kb, up to about 14 kb, or up to about 15 kb. In some embodiments, a construct is a plasmid and can have a total length in a range of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 1 kb to about 9 kb, about 1 kb to about 10 kb, about 1 kb to about 11 kb, about 1 kb to about 12 kb, about 1 kb to about 13 kb, about 1 kb to about 14 kb, or about 1 kb to about 15 kb.
[0127] In some embodiments, a construct is a viral construct and can have a total number of nucleotides of up to 10 kb. In some embodiments, a viral construct can have a total number of nucleotides in the range of about 1 kb to about 2 kb, 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 1 kb to about 9 kb, about 1 kb to about 10 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 2 kb to about 9 kb, about 2 kb to about 10 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 3 kb to about 9 kb, about 3 kb to about 10 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 4 kb to about 9 kb, about 4 kb to about 10 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 5 kb to about 9 kb, about 5 kb to about 10 kb, about 6 kb to about 7 kb, about 6 kb to about 8 kb, about 6 kb to about 9 kb, about 6 kb to about 10 kb, about 7 kb to about 8 kb, about 7 kb to about 9 kb, about 7 kb to about 10 kb, about 8 kb to about 9 kb, about 8 kb to about 10 kb, or about 9 kb to about 10 kb.
[0128] In some embodiments, a construct is a lentivirus construct and can have a total number of nucleotides of up to 8 kb. In some examples, a lentivirus construct can have a total number of nucleotides of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb, or about 7 kb to about 8 kb
[0129] In some embodiments, a construct is an adenovirus construct and can have a total number of nucleotides of up to 8 kb. In some embodiments, an adenovirus construct can have a total number of nucleotides in the range of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 6 kb to about 7 kb, about 6 kb to about 8 kb, or about 7 kb to about 8 kb.
[0130] AAV for Therapeutic Uses
[0131] Previous research has identified Adeno Associate Virus (AAV) as a suitable vector for the delivery of therapeutic oligonucleotides to subjects in need thereof.
[0132] Gene therapy is evolving rapidly for the treatment of genetic disorders, and for many patients, the success of these therapeutics is their only hope for a cure (Angeula & High 2019; and Carlton 2018). During gene therapy, a healthy copy of the gene causing the disease is delivered to the patient via a viral vector (Angeula & High, 2019; and Carlton, 2018). Many regulatory bodies (EMA, FDA, etc.) require monitoring of the shed virus as part of the gene therapy clinical trial (FDA, 2015; ICH, 2009; GTWP, BWP, and SWP, 2008; and Bubela et al., 2019). Detection of the virus in bodily fluids might be critical to understand environmental consequences or potential long-term effects that may lead to an increase of neutralizing antibodies (NAb) against adeno-associated virus in society (Bubela et al., 2019; and Rodrigues et al., 2018).
[0133] One of the most common gene therapy vectors is adeno-associated virus (AAV). AAV is used due to its apparent lack of pathogenesis and replication competency (Angeula & High 2019; and Carlton 2018). Patients undergoing recombinant AAV (rAAV) gene therapy treatment are injected with high titers of rAAV. Patients undergoing this kind of treatment clear excess virus through a process called shedding. Shedding refers to the excretion or release of the vector-based gene therapy product from patients' excreta (stool), secreta (urine, saliva, and semen), or blood products (whole blood, plasma, or PBMCs) (FDA, 2015; ICH, 2009; GTWP, BWP, and SWP, 2008; and Bubela et al., 2019). Even though AAV is not pathogenic, shedding raises a possible risk to people encountering patients undergoing gene therapy treatment. The risk of exposure to the virus might result in increase of neutralizing antibodies against AAVs and rAAV-based therapy in society (Bubela et al., 2019; and Rodrigues et al., 2018). Monitoring viral concentration in shed patients' samples is necessary to understand possible modes of vector transmission and is a requirement of multiple regulatory agencies.
[0134] There are two key requirements for developing a shedding assay: The assay must be developed for a diverse range of sample types, and must be quantitative (FDA, 2015). Herein are described novel compositions comprising specific primers to quantify viral DNA in patient samples (
[0135] Described herein are new compositions and protocols for monitoring viral-shed DNA from many different bodily fluids, including semen, saliva, urine, whole blood, PBMC, plasma, and stool. The data presented represents an optimized quantification protocol from a diverse cohort of shedding compartments to establish consistent and reproducible shedding assays.
Viral Shedding
[0136] Existing DNA quantification and/or sequencing materials and methods for accurately detecting, identifying, and quantifying foreign and/or therapeutic oligonucleotides are often found wanting. In particular, current materials and methods for quantification of gene therapy constructs in clinical samples and/or shed from patients often fail to accurately and reproducibly quantify the shed construct and/or genome titers to the levels described by the appropriate regulatory and/or advisory bodies (e.g., the GTWP, BWP, SWP and/or FDA).
[0137] In some embodiments, the materials and methods described herein can detect and quantify gene therapy constructs found as unbound and/or free oligonucleotides. In some embodiments, the materials and methods described herein can detect and quantify gene therapy constructs associated with a viral particle. As described herein, there are many suitable gene therapy vectors (e.g., viral particles) that may house gene therapy oligonucleotides amenable to quantification using the materials and methods described herein. In certain embodiments, a gene therapy construct detected and quantified is an oligonucleotide associated with a recombinant adeno-associated virus (rAAV) particle.
[0138] Previous assays have not demonstrated sufficient performance to be widely accepted in clinical laboratories and have not been submitted for regulatory approval. Most are used as research assays or local home-brew laboratory developed tests due to these performance limitations. Previous assay designs have not solved these limitations.
[0139] The present disclosure, in contrast, results in uniquely high performing assay design prototypes. The top performing design meets IVD commercialization criteria of >95% successful amplification with known global sequence variants. This performance criteria has been previously demonstrated to be indicative of sufficient sensitivity across genetic variants to be implemented for routine clinical use with samples in international studies.
Measuring Viral Associated Oligonucleotide Construct Titers from Biological Sources
[0140] In some embodiments, the compositions and methods described herein are useful for detecting and quantifying oligonucleotide constructs delivered by rAAV particle. In some embodiments, the detection and quantification is conducted on samples obtained from a patient and are measurements of viral shedding. In some embodiments, the viral associated oligonucleotide constructs shed from a patient may be direct to a specific gene therapy construct, such as but not limited to constructs for treating: Duchenne Muscular Dystrophy, and/or Hemophilia B.
Amplification Oligonucleotide Sequences and Primer Sets
[0141] In some embodiments, compositions described herein comprise forward and reverse oligonucleotides suitable and specific for amplification and subsequent quantification of known gene therapy constructs. In some embodiments, compositions comprising forward and reverse amplification oligonucleotides may additionally comprise sequence specific probes suitable for real time quantification of amplicon products. In some embodiments, sequence specific probes may be probes designed for amplicon quantification using TaqMan™ quantitative polymerase chain reaction (qPCR) protocols.
[0142] In some embodiments, quantification probes and primers may be designed to hybridize with sequences specific to non-naturally occurring construct specific junctions. In some embodiments, junctions may be spanning construct-to-regulatory elements, (e.g., spanning rAAV inverted terminal repeat sequences to promoter and/or 3′ UTR regions). In some embodiments, junctions may be spanning regulatory-to-regulatory elements, (e.g., spanning a promoter to enhancer junction, and/or spanning a 3′ UTR to polyA signal sequence). In some embodiments, junctions may be spanning regulatory-to-payload elements (e.g., spanning promoter and/or 3′ UTR regions to gene therapy specific payload sequences, i.e., Dystrophin, Mini-Dystrophin, and/or Factor IX). In some embodiments, junctions may be spanning construct-to-payload elements (e.g., spanning rAAV inverted terminal repeat sequences to gene therapy specific payload sequences).
[0143] In some embodiments, primers and/or probes may be screened to determine the total pool of primers and/or probes amenable to multiplexing. In some embodiments, one or more primers and/or one or more probes may be utilized in a multiplexing assay. In certain embodiments, primers and/or probes are screened for crosstalk and/or undesirable oligonucleotide interactions. In some embodiments, primers and/or probes are tested to evaluate their specificity when additionally exposed to background genomic DNA. In some embodiments, candidate primers and/or probes are further optimized to include additions, truncations, and/or nucleotide modifications to increase assay performance. In certain embodiments, primers and/or probes tested in numerous different concentrations (e.g., 100 nM, 200M, 300 nM, 400 nM, and/or 500 nM) to increase assay performance. In some embodiments, specific primer and/or probe combinations may be utilized to increase assay performance. In some embodiments, any or all of the factors described herein may be utilized in determining an appropriate primer and/or primer and probe set for the accurate amplification and/or quantification of an AAV construct.
[0144] In some embodiments, an active fragment of an oligonucleotide described herein is at most the length of a particular primer and/or probe minus one nucleotide. In some embodiments, an active fragment is at most 29 nucleotides, is at most 28 nucleotides, is at most 27 nucleotides, is at most 26 nucleotides, is at most 25 nucleotides, is at most 24 nucleotides, is at most 23 nucleotides, is at most 22 nucleotides, is at most 21 nucleotides, is at most 20 nucleotides, is at most 19 nucleotides, is at most 18 nucleotides, is at most 17 nucleotides, is at most 16 nucleotides, is at most 15 nucleotides, is at most 14 nucleotides, is at most 13 nucleotides, is at most 12 nucleotides, is at most 11 nucleotides, or is at most 10 nucleotides.
[0145] In some embodiments, primer combinations and/or primer and probe combinations may be multiplexed with additional primer and/or primer and probe sets. In some embodiments, the sensitivity of a primer combination and/or primer and probe combination may be altered with multiplexing (e.g., if CTs of single assays come up much earlier than in multiplex assay, then multiplexing may have an inhibitory effect and this may alter the overall sensitivity). In some embodiments, the specificity of a primer combination and/or primer and probe combination may be altered with multiplexing (e.g., if in a multiplex assay wherein the primers and/or primer and probe combinations are given multiple target DNA populations as one template, a specific primer combinations and/or primer and probe combinations may not produce signal in the absence of its target DNA population, if signal is present, specificity may be lower than in the absence of the additional DNA population).
[0146] Oligonucleotide Sequences for DMD AAV Specific Quantification
[0147] Each of the following Primer sequences is provided in 5′ to 3′ order; those skilled in the art will recognize than in certain embodiments a polynucleotide may be an RNA molecule, or a DNA molecule. In certain embodiments, primary screening comprised the evaluation of 37 primer combinations and 9 different probes, culminating in 40 different primer-probe combinations.
TABLE-US-00002 DMD-FWD-A SEQ ID NO: 1 AGA CAG ACA CTC AGG AGC CAG CC DMD-FWD-A.U SEQ ID NO: 2 AGA CAG ACA CUC AGG AGC CAG CC DMD-FWD-B SEQ ID NO: 3 ACC ACC TCC ACA GCA CAG ACA GA DMD-FWD-B.U SEQ ID NO: 4 ACC ACC UCC ACA GCA CAG ACA GA DMD-FWD-C SEQ ID NO: 5 CCT ACT ACA TCA ACC ACG AGA CC DMD-FWD-C.U SEQ ID NO: 6 CCU ACU ACA UCA ACC ACG AGA CC DMD-FWD-D SEQ ID NO: 7 GGA TAA GTA CCG CTA CCT GTT CA DMD-FWD-D.U SEQ ID NO: 8 GGA UAA GUA CCG CUA CCU GUU CA DMD-REV-A SEQ ID NO: 9 AGC AGT CCT CCA CTT CCT CCC AC DMD-REV-A.U SEQ ID NO: 10 AGC AGU CCU CCA CUU CCU CCC AC DMD-REV-B SEQ ID NO: 11 TGC ACG TCC TCT CTC TCG TAG CA DMD-REV-B.U SEQ ID NO: 12 UGC ACG UCC UCU CUC UCG UAG CA DMD-REV-C SEQ ID NO: 13 CTA GGG ATC TGG ATG CTA TCG TG DMD-REV-C.U SEQ ID NO: 14 CUA GGG AUC UGG AUG CUA UCG UG DMD-REV-D SEQ ID NO: 15 CTC TGA TAC AGC TCG GTC ATC TT DMD-REV-D.U SEQ ID NO: 16 CUC UGA UAC AGC UCG GUC AUC UU DMD-PROBE-A SEQ ID NO: 17 /56-FAM/AGC GTC GAG /ZEN/CGG CCG ATC CGC CAC C/3IABkFQ/ DMD-PROBE-A.U SEQ ID NO: 18 /56-FAM/AGC GUC GAG /ZEN/CGG CCG AUC CGC CAC C/3IABkFQ/ DMD-PROBE-A.0 SEQ ID NO: 19 AGC GTC GAG CGG CCG ATC CGC CAC C DMD-PROBE-A.0.U SEQ ID NO: 20 AGC GUC GAG CGG CCG AUC CGC CAC C DMD-PROBE-B SEQ ID NO: 21 /56-FAM/CAC CAA AGC /ZEN/ ATG GTG GCG GAT CG /3IABkFQ/ DMD-PROBE-B.U SEQ ID NO: 22 /56-FAM/CAC CAA AGC /ZEN/ AUG GUG GCG GAU CG /3IABkFQ/ DMD-PROBE-B.0 SEQ ID NO: 23 CAC CAA AGC ATG GTG GCG GAT CG DMD-PROBE-B.0.U SEQ ID NO: 24 CAC CAA AGC AUG GUG GCG GAU CG DMD-PROBE-C SEQ ID NO: 25 /56-FAM/ATC AGA GGA /ZEN/GAC TGG GCC TGC TGC T /3IABkFQ/ DMD-PROBE-C.U SEQ ID NO: 26 /56-FAM/AUC AGA GGA /ZEN/GAC UGG GCC UGC UGC U /3IABkFQ/ DMD-PROBE-C.0 SEQ ID NO: 27 ATC AGA GGA GAC TGG GCC TGC TGC T DMD-PROBE-C.0.U SEQ ID NO: 28 AUC AGA GGA GAC UGG GCC UGC UGC U DMD-PROBE-D SEQ ID NO: 29 /56-FAM/CAG ACC ACC /ZEN/ TGC TGG GAC CAC CCT /3IABkFQ/ DMD-PROBE-D.U SEQ ID NO: 30 /56-FAM/CAG ACC ACC /ZEN/ UGC UGG GAC CAC CCU /3IABkFQ/ DMD-PROBE-D.0 SEQ ID NO: 31 CAG ACC ACC TGC TGG GAC CAC CCT DMD-PROBE-D.0.U SEQ ID NO: 32 CAG ACC ACC UGC UGG GAC CAC CCU DMD-FWD-E SEQ ID NO: 63 CAG GAT GGG CTA CCT GCC CGT G DMD-FWD-E.U SEQ ID NO: 64 CAG GAU GGG CUA CCU GCC CGU G DMD-FWD-F SEQ ID NO: 65 CCT ACT ACA TCA ACC ACG AGA CC DMD-FWD-F.U SEQ ID NO: 66 CCU ACU ACA UCA ACC ACG AGA CC DMD-REV-E SEQ ID NO: 67 CAC TCT GAT CGA TGC ATC TGA GCT CTT DMD-REV-E.U SEQ ID NO: 68 CAC UCU GAU CGA UGC AUC UGA GCU CUU DMD-REV-F SEQ ID NO: 69 TAG GCC TCT CGA GCT CCT CAT CA DMD-REV-F.U SEQ ID NO: 70 UAG GCC UCU CGA GCU CCU CAU CA DMD-REV-G SEQ ID NO: 71 CTC TGA TAC AGC TCG GTC ATC TT DMD-REV-G.U SEQ ID NO: 72 CUC UGA UAC AGC UCG GUC AUC UU DMD-PROBE-F SEQ ID NO: 73 /56-FAM/ACC GTG CTG /ZEN/ GAA GGC GAC AAC ATG GAG ACC /3IABkFQ/ DMD-PROBE-F.U SEQ ID NO: 74 /56-FAM/ACC GUG CUG /ZEN/ GAA GGC GAC AAC AUG GAG ACC /3IABkFQ/ DMD-PROBE-F.0 SEQ ID NO: 75 ACC GTG CTG GAA GGC GAC AAC ATG GAG ACC DMD-PROBE-F.0.U SEQ ID NO: 76 ACC GUG CUG GAA GGC GAC AAC AUG GAG ACC DMD-PROBE-G SEQ ID NO: 77 /56-FAM/CAG ACC ACC /ZEN/ TGC TGG GAC CAC CCT /3IABkFQ/ DMD-PROBE-G.U SEQ ID NO: 78 /56-FAM/CAG ACC ACC /ZEN/ UGC UGG GAC CAC CCU /3IABkFQ/ DMD-PROBE-G.0 SEQ ID NO: 79 CAG ACC ACC TGC TGG GAC CAC CCT DMD-PROBE-G.0.U SEQ ID NO: 80 CAG ACC ACC UGC UGG GAC CAC CCU
[0148] Oligonucleotide Combinations for DMD AAV Specific Quantification
[0149] In some embodiments, primer combinations may be utilized for accurate and specific DMD AAV quantification.
[0150] In some embodiments, a combination of primers can comprise one or more forward primers. In some embodiments, a combination of primers can comprise one or more reverse primers. In some embodiments, one or more forward primers can comprise or consist of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, or a combination thereof. In some embodiments, one or more reverse primers can comprise or consist of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, or a combination thereof.
[0151] In some embodiments, a composition comprises a combination of primers and one or more probes. In some embodiments, one or more probes can comprise or consist of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, or a combination thereof.
[0152] In some embodiments, the primer combination comprises DMD-FWD-A (SEQ ID NO: 1) and DMD-REV-B (SEQ ID NO: 11). In some embodiments, the primer combination comprising DMD-FWD-A (SEQ ID NO: 1) and DMD-REV-B (SEQ ID NO: 11) is coupled with the probe sequence DMD-PROBE-A (SEQ ID NO: 17). In some embodiments, the primer combination comprising DMD-FWD-A (SEQ ID NO: 1) and DMD-REV-B (SEQ ID NO: 11) is coupled with the probe sequence DMD-PROBE-B (SEQ ID NO: 21).
[0153] In some embodiments, the primer combination comprises DMD-FWD-B (SEQ ID NO: 3), and DMD-REV-A (SEQ ID NO: 9). In some embodiments, the primer combination comprising DMD-FWD-B (SEQ ID NO: 3), and DMD-REV-A (SEQ ID NO: 9) is coupled with the probe sequence DMD-PROBE-A (SEQ ID NO: 17).
[0154] In some embodiments, the primer combination comprises DMD-FWD-E (SEQ ID NO: 63) and DMD-REV-E (SEQ ID NO: 67). In some embodiments, the primer combination comprising DMD-FWD-E (SEQ ID NO: 63) and DMD-REV-E (SEQ ID NO: 67) is coupled with the probe sequence DMD-PROBE-F (SEQ ID NO: 73).
[0155] In some embodiments, the primer combination comprises DMD-FWD-F (SEQ ID NO: 69) and DMD-REV-G (SEQ ID NO: 71). In some embodiments, the primer combination comprising DMD-FWD-F (SEQ ID NO: 69) and DMD-REV-G (SEQ ID NO: 71) is coupled with the probe sequence DMD-PROBE-G (SEQ ID NO: 77).
[0156] In some embodiments, the primer combination comprises DMD-FWD-B (SEQ ID NO:3) and DMD-REV-A (SEQ ID NO: 9). In some embodiments, the primer combination comprising DMD-DMD-FWD-B (SEQ ID NO:3) and DMD-REV-A (SEQ ID NO: 9) is coupled with the probe sequence DMD-PROBE-A (SEQ ID NO: 17).
[0157] In some embodiments, the primer combination comprises DMD-FWD-C (SEQ ID NO: 5) and DMD-REV-D (SEQ ID NO: 15). In some embodiments, the primer combination comprising DMD-FWD-C(SEQ ID NO: 5) and DMD-REV-D (SEQ ID NO: 15) is coupled with the probe sequence DMD-PROBE-D (SEQ ID NO: 29).
[0158] In some embodiments, the primer combination comprises DMD-FWD-D (SEQ ID NO: 7), and DMD-REV-C(SEQ ID NO: 13). In some embodiments, the primer combination comprising DMD-FWD-D (SEQ ID NO: 7), and DMD-REV-C(SEQ ID NO: 13) is coupled with the probe sequence DMD-PROBE-C(SEQ ID NO: 25).
[0159] Oligonucleotide Sequences for HEM-B AAV Specific Quantification
[0160] Each of the following Primer sequences is provided in 5′ to 3′ order; those skilled in the art will recognize than in certain embodiments a polynucleotide may be an RNA molecule, or a DNA molecule. In certain embodiments, primary screening comprised the evaluation of 72 primer combinations and 5 different probes, culminating in 47 different primer-probe combinations.
TABLE-US-00003 HemB-FWD-A SEQ ID NO: 33 GTG GAG AGG AGC AGA GGT TGT C HemB-FWD-A.U SEQ ID NO: 34 GUG GAG AGG AGC AGA GGU UGU C HemB-FWD-B SEQ ID NO: 35 CAC TGC TTA AAT ACG GAC GAG GA HemB-FWD-B.U SEQ ID NO: 36 CAC UGC UUA AAU ACG GAC GAG GA HemB-FWD-C SEQ ID NO: 37 GAG GCA CCA CCA CTG ACC T HemB-FWD-C.U SEQ ID NO: 38 GAG GCA CCA CCA CUG ACC U HemB-REV-A SEQ ID NO: 39 CTG TTC CAC TGG TAG CAA GAT CC HemB-REV-A.U SEQ ID NO: 40 GUG UUC CAC UGG UAG CAA GAU CC HemB-REV-B SEQ ID NO: 41 ATG ATC ATG TTC ACC CTC TGC AT HemB-REV-B.U SEQ ID NO: 42 AUG AUC AUG UUC ACC CUC UGC AU HemB-REV-C SEQ ID NO: 43 CAT AAC CTT TGC TAG CAG ATT GTG HemB-REV-C.U SEQ ID NO: 44 CAU AAC CUU UGC UAG CAG AUU GUG ACG HemB-REV-D SEQ ID NO: 45 CTT TGC TAG GAG ATT GTG AAA GTG HemB-REV-D.U SEQ ID NO: 46 CXX XGC XAG CAG AXX GXG AAA GXG HemB-PROBE-A SEQ ID NO: 47 /56-FAM/CCC TCT CAC /ZEN/ACT ACC TAA ACC ACG CCA /3IABkFQ/ HemB-PROBE-A.U SEQ ID NO: 48 /56-FAM/CCC UCU CAC /ZEN/ACU ACC UAA ACC GGC C/3IABkFQ/ HemB-PROBE-A.0 SEQ ID NO: 49 CCC TCT CAC ACT ACC TAA ACC ACG CCA HemB-PROBE-A.0.U SEQ ID NO: 50 CCC UCU CAC ACU ACC UAA ACC ACG CCA HemB-PROBE-B SEQ ID NO: 51 /56-FAM/TGC CTG AAG /ZEN/CTG AGG AGA CAG HemB-PROBE-B.U GGC C/3IABkFQ/ SEQ ID NO: 52 /56-FAM/UGC CUG AAG /ZEN/CUG AGG AGA CAG GGA C/3IABkFQ/ HemB-PROBE-B.0 SEQ ID NO: 53 TGC CTG AAG CTG AGG AGA CAG GGC C HemB-PROBE-B.0.U SEQ ID NO: 54 UGC CUG AAG CUG AGG AGA CAG GGC C HemB-PROBE-C SEQ ID NO: 55 /56-FAM/TCA GGC ACC /ZEN/ACC ACT GAC CTG GGA C/3IABkFQ/ HemB-PROBE-C.U SEQ ID NO: 56 /56-FAM/UCA GGC ACC /ZEN/ACC ACU GAC CUG HemB-PROBE-C.0 SEQ ID NO: 57 TCA GGC ACC ACC ACT GAC CTG GGA C HemB-PROBE-C.0.U SEQ ID NO: 58 UCA GGC ACC ACC ACU GAC CUG GGA C HemB-PROBE-D SEQ ID NO: 59 /56-FAM/AGC AGA TTG /ZEN/TGA AAG TGG TAT TCA CTG TCC /3IABkFQ/ HemB-PROBE-D.U SEQ ID NO: 60 /56-FAM/AGC AGA UUG /ZEN/UGA AAG UGG UAU UCA CUG UCC /3IABkFQ/ HemB-PROBE-D.0 SEQ ID NO: 61 AGC AGA TTG TGA AAG TGG TAT TCA CTG TCC HemB-PROBE-D.0.U SEQ ID NO: 62 AGC AGA UUG UGA AAG UGG UAU UCA CUG UCC HemB-FWD-D SEQ ID NO: 81 ACT GGA TTA AGG AGA AAA CCA AGC TG HemB-FWD-D.U SEQ ID NO: 82 ACU GGA UUA AGG AGA AAA CCA AGC UG HemB-FWD-E SEQ ID NO: 83 TGT GAA CTG GAT TAA GGA GAA AAC CA HemB-FWD-E.U SEQ ID NO: 84 UGU GAA CUG GAU UAA GGA GAA AAC CA HemB-REV-E SEQ ID NO: 85 TCT ATA TCT AAA AGG CAA GCA TAG TCA HemB-REV-E.U SEQ ID NO: 86 UCU AUA UCU AAA AGG CAA GCA UAC UCA HemB-REV-F SEQ ID NO: 87 GGC AAC TAG AAG GCA CAG TAG A HemB-REV-E.U SEQ ID NO: 88 GGC AAC UAG AAG GCA CAG UAG A HemB-PROBE-E SEQ ID NO: 89 /56-FAM/CTG CAG CCA /ZEN/ GGG GGA TCA GCC /3IABkFQ/ HemB-PROBE-E.U SEQ ID NO: 90 /56-EAM/CUG CAG CCA /ZEN/ GGG GGA UCA GCC /3IABkFQ/ HemB-PROBE-E.0 SEQ ID NO: 91 CTG CAG CCA GGG GGA TCA GCC HemB-PROBE-E.0.U SEQ ID NO: 92 CUG CAG CCA GGG GGA UCA GCC
[0161] Oligonucleotide Combinations for HEM-B AAV Specific Quantification
[0162] In some embodiments, primer combinations may be utilized for accurate and specific Hem-B AAV quantification.
[0163] In some embodiments, a combination of primers can comprise one or more forward primers. In some embodiments, a combination of primers can comprise one or more reverse primers. In some embodiments, one or more forward primers can comprise or consist of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, or a combination thereof. In some embodiments, one or more reverse primers can comprise or consist of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, or a combination thereof.
[0164] In some embodiments, a composition comprises a combination of primers and one or more probes. In some embodiments, one or more probes can comprise or consist of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 91, SEQ ID NO: 92, or a combination thereof.
[0165] In some embodiments, the primer combination comprises HemB-FWD-B (SEQ ID NO: 35) and HemB-REV-D (SEQ ID NO: 45). In some embodiments, the primer combination comprising HemB-FWD-B (SEQ ID NO: 35) and HemB-REV-D (SEQ ID NO: 45) is coupled with the probe sequence HemB-PROBE-C(SEQ ID NO: 55).
[0166] In some embodiments, the primer combination comprises HemB-FWD-A (SEQ ID NO: 33) and HemB-REV-A (SEQ ID NO: 39). In some embodiments, the primer combination comprising HemB-FWD-A (SEQ ID NO: 33) and HemB-REV-A (SEQ ID NO: 39) is coupled with the probe sequence HemB-PROBE-A (SEQ ID NO: 47).
[0167] In some embodiments, the primer combination comprises HemB-FWD-D (SEQ ID NO: 81) and HemB-REV-F (SEQ ID NO: 87). In some embodiments, the primer combination comprising HemB-FWD-D (SEQ ID NO: 81) and HemB-REV-F (SEQ ID NO: 87) is coupled with the probe sequence HemB-PROBE-E (SEQ ID NO:89).
[0168] In some embodiments, the primer combination comprises HemB-FWD-B (SEQ ID NO: 35) and HemB-REV-C(SEQ ID NO: 43). In some embodiments, the primer combination comprising HemB-FWD-B (SEQ ID NO: 35) and HemB-REV-C(SEQ ID NO: 43) is coupled with the probe sequence HemB-PROBE-B (SEQ ID NO: 51).
[0169] In some embodiments, the primer combination comprises HemB-FWD-C (SEQ ID NO: 37) and HemB-REV-B (SEQ ID NO: 41). In some embodiments, the primer combination comprising HemB-FWD-C(SEQ ID NO: 37) and HemB-REV-B (SEQ ID NO: 41) is coupled with the probe sequence HemB-PROBE-D (SEQ ID NO: 59).
[0170] In some embodiments, the primer combination comprises HemB-FWD-C (SEQ ID NO: 37) and HemB-REV-B (SEQ ID NO: 41). In some embodiments, the primer combination comprising HemB-FWD-C(SEQ ID NO: 37) and HemB-REV-B (SEQ ID NO: 41) is coupled with the probe sequence HemB-PROBE-D (SEQ ID NO: 59).
[0171] In some embodiments, the primer combination comprises HemB-FWD-E (SEQ ID NO: 83) and HemB-REV-F (SEQ ID NO: 85). In some embodiments, the primer combination comprising HemB-FWD-E (SEQ ID NO: 83) and HemB-REV-F (SEQ ID NO: 85) is coupled with the probe sequence HemB-PROBE-E (SEQ ID NO: 89).
Oligonucleotide Preparation
[0172] Oligonucleotides of the present disclosure may be prepared by any of a variety of methods (see, e.g., Sambrook et al., “Molecular Cloning: A Laboratory Manual”, 1989, 2.sup.nd Ed., Cold Spring Harbour Laboratory Press: New York, N.Y.; “PCR Protocols: A Guide to Methods and Applications”, 1990, Innis (Ed.), Academic Press: New York, N.Y.; Tijssen “Hybridization with Nucleic Acid Probes—Laboratory Techniques in Biochemistry and Molecular Biology (Parts I and II)”, 1993, Elsevier Science; “PCR Strategies”, 1995, Innis (Ed.), Academic Press: New York, N.Y.; and “Short Protocols in Molecular Biology”, 2002, Ausubel (Ed.), 5.sup.th Ed., John Wiley & Sons: Secaucus, N.J.).
[0173] In some embodiments, oligonucleotides may be prepared by chemical techniques well-known in the art, including, e.g., chemical synthesis and polymerization based on a template as described, e.g., in Narang et al., Meth. Enzymol. 68:90-98 (1979); Brown et al., Meth. Enzymol. 68: 109-151 (1979); Belousov et al., Nucleic Acids Res. 25:3440-3444 (1997); Guschin et al., Anal. Biochem. 250:203-211 (1997); Blommers et al., Biochemistry 33:7886-7896 (1994); Frenkel et al., Free Radic. Biol. Med. 19:373-380 (1995); and U.S. Pat. No. 4,458,066.
[0174] In some embodiments, oligonucleotides may be prepared using an automated, solid-phase procedure based on the phosphoramidite approach. In such methods, each nucleotide is individually added to the 5′-end of the growing oligonucleotide chain, which is attached at the 3′-end to a solid support. The added nucleotides are in the form of trivalent 3′-phosphoramidites that are protected from polymerization by a dimethoxytriyl (or DMT) group at the 5′-position. After base-induced phosphoramidite coupling, mild oxidation to give a pentavalent phosphotriester intermediate and DMT removal provides a new site for oligonucleotide elongation. The oligonucleotides are then cleaved off the solid support, and the phosphodiester and exocyclic amino groups are deprotected with ammonium hydroxide. These syntheses may be performed on oligo synthesizers such as those commercially available from Perkin Elmer/Applied Biosystems, Inc. (Foster City, Calif.), DuPont (Wilmington, Del.) or Milligen (Bedford, Mass.). Alternatively, oligonucleotides can be custom made and ordered from a variety of commercial sources well-known in the art, including, for example, the Midland Certified Reagent Company (Midland, Tex.), ExpressGen, Inc. (Chicago, Ill.), Operon Technologies, Inc. (Huntsville, Ala.), and many others.
[0175] Purification of oligonucleotides, where necessary or desirable, may be carried out by any of a variety of methods well-known in the art. For example, purification of oligonucleotides is typically performed either by native acrylamide gel electrophoresis, by anion-exchange HPLC, e.g., see Pearson and Regnier, J. Chrom. 255:137-149 (1983) or by reverse phase HPLC, e.g., see McFarland and Borer, Nucleic Acids Res. 7:1067-1080 (1979).
[0176] The sequence of oligonucleotides can be verified using any suitable sequencing method including, but not limited to, chemical degradation, e.g., see Maxam and Gilbert, Methods of Enzymology, 65:499-560 (1980), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, e.g., see Pieles et al., Nucleic Acids Res. 21:3191-3196 (1993), mass spectrometry following a combination of alkaline phosphatase and exonuclease digestions, e.g., see Wu and Aboleneen, Anal. Biochem. 290:347-352 (2001).
[0177] The present disclosure encompasses modified versions of these oligonucleotides that perform as equivalents of these oligonucleotides in accordance with the methods of the present disclosure. These modified oligonucleotides may be prepared using any of several means known in the art. Non-limiting examples of such modifications include methylation, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.), or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Modified oligonucleotide may also be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Furthermore, the oligonucleotides of the present disclosure may also be modified with a label.
Labeling of Oligonucleotides
[0178] In some embodiments, the primers are labeled with a detectable agent or moiety before being used in amplification/detection assays. The role of a detectable agent is to allow visualization and detection of amplified target sequences. Preferably, the detectable agent is selected such that it generates a signal which can be measured and whose intensity is related (e.g., proportional) to the amount of amplification products in the sample being analyzed.
[0179] The association between the oligonucleotide and the detectable agent can be covalent or non-covalent. Labeled detection primers can be prepared by incorporation of or conjugation to a detectable moiety. Labels can be attached directly to the nucleic acid sequence or indirectly (e.g., through a linker). Linkers or spacer arms of various lengths are known in the art and are commercially available, and can be selected to reduce steric hindrance, or to confer other useful or desired properties to the resulting labeled molecules, e.g., see Mansfield et al., Mol. Cell Probes 9:145-156 (1995).
[0180] Various methods for labeling nucleic acid molecules are known in the art. For a review of labeling protocols, label detection techniques, and recent developments in the field, see, for example, Kricka, Ann. Clin. Biochem. 39:114-129 (2002); van Gijlswijk et al., Expert Rev. Mol. Diagn. 1:81-91 (2001); and Joos et al., J. Biotechnol. 35:135-153 (1994). Standard nucleic acid labeling methods include: incorporation of radioactive agents, direct attachments of fluorescent dyes (Smith et al., Nucl. Acids Res. 13:2399-2412 (1985)) or of enzymes (Connoly and Rider, Nucl. Acids. Res. 13:4485-4502 (1985)); chemical modifications of nucleic acid molecules making them detectable immunochemically or by other affinity reactions, e.g., see Broker et al., Nucl. Acids Res. 5:363-384 (1978); Bayer et al., Methods of Biochem. Analysis 26:1-45 (1980); Langer et al., Proc. Natl. Acad. Sci. USA 78:6633-6637 (1981); Richardson et al., Nucl. Acids Res. 11:6167-6184 (1983); Brigati et al., Virol. 126:32-50 (1983); Tchen et al., Proc. Natl. Acad. Sci. USA 81:3466-3470 (1984); Landegent et al., Exp. Cell Res. 15:61-72 (1984); and Hopman et al., Exp. Cell Res. 169:357-368 (1987); and enzyme-mediated labeling methods, such as random priming, nick translation, PCR and tailing with terminal transferase. For a review on enzymatic labeling, see, e.g., Temsamani and Agrawal, Mol. Biotechnol. 5:223-232 (1996). More recently developed nucleic acid labeling systems include, but are not limited to: ULS (Universal Linkage System), which is based on the reaction of monoreactive cisplatin derivatives with the N7 position of guanine moieties in DNA (Heetebrij et al., Cytogenet. Cell. Genet. 87:47-52 (1999)), psoralen-biotin, which intercalates into nucleic acids and upon UV irradiation becomes covalently bonded to the nucleotide bases (Levenson et al., Methods Enzymol. 184:577-583 (1990); and Pfannschmidt et al., Nucleic Acids Res. 24:1702-1709 (1996)), photoreactive azido derivatives (Neves et al., Bioconjugate Chem. 11:51-55 (2000)), and DNA alkylating agents (Sebestyen et al., Nat. Biotechnol. 16: 568-576 (1998)).
[0181] It will be appreciated that any of a wide variety of detectable agents can be used in the practice of the present disclosure. Suitable detectable agents include, but are not limited to, various ligands, radionuclides (such as, for example, .sup.32P, .sup.35S, .sup.3H, .sup.14C, .sup.125I, .sup.131I and the like); fluorescent dyes (such as, for example, FAM, Yakima Yellow®, SUN™, HEX, Cy® 3, Texas Red®-X, and/or Cy® 5); fluorescent dye quenchers (such as, for example, ZEN, Iowa Black™ FQ, Iowa Black RQ, and/or TAO), chemiluminescent agents (such as, for example, acridinium esters, stabilized dioxetanes, and the like); spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper and platinum) or nanoclusters; enzymes (such as, for example, those used in an ELISA, e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase); colorimetric labels (such as, for example, dyes, colloidal gold, and the like); magnetic labels (such as, for example, Dynabeads™); and biotin, dioxigenin or other haptens and proteins for which antisera or monoclonal antibodies are available.
[0182] A “tail” of normal or modified nucleotides can also be added to tag an oligonucleotide for detectability purposes. In some embodiments, an M13 tag sequence may be added.
Extraction and Preparation of Viral Oligonucleotides from Biological Samples
[0183] In some embodiments, materials and methods of the present disclosure may include oligonucleotides extracted and prepared from biological samples. In some embodiments, suitable biological samples for the extraction of oligonucleotides include but are not limited to: urine, semen, plasma, stool, whole blood, and/or saliva. The extraction and preparation of oligonucleotides from biological samples occurs through methods generally known in the art (e.g., VERSANT® kPCR Sample Preparation 1.0 and/or 1.2).
Amplification Methods and Reactions
[0184] In some embodiments, the present disclosure provides methods that use the aforementioned oligonucleotides as amplification primers to amplify regions of specific AAV constructs, in particular regions that are not found in natural AAV, and are specific to certain therapeutic AAVs. As discussed in more detail below, in some embodiments the primers are used in quantitative PCR methods for the amplification and detection of specific AAV constructs.
[0185] The use of oligonucleotide sequences of the present disclosure as primers to amplify AAV target sequences in test samples is not limited to any particular nucleic acid amplification technique or any particular modification thereof. In fact, the inventive oligonucleotide sequences can be employed in any of a variety of nucleic acid amplification methods well-known in the art (see, for example, Kimmel and Berger, Methods Enzymol. 152: 307-316 (1987); Sambrook et al., “Molecular Cloning: A Laboratory Manual”, 1989, 2.sup.nd Ed., Cold Spring Harbour Laboratory Press: New York, N.Y.; “Short Protocols in Molecular Biology”, Ausubel (Ed.), 2002, 5.sup.th Ed., John Wiley & Sons: Secaucus, N.J.).
[0186] Such nucleic acid amplification methods include, but are not limited to, the Polymerase Chain Reaction (or PCR, described, for example, in “PCR Protocols: A Guide to Methods and Applications”, Innis (Ed.), 1990, Academic Press: New York; “PCR Strategies”, Innis (Ed.), 1995, Academic Press: New York; “Polymerase chain reaction: basic principles and automation in PCR: A Practical Approach”, McPherson et al. (Eds.), 1991, IRL Press: Oxford; Saiki et al., Nature 324:163 (1986); and U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,889,818, each of which is incorporated herein by reference in its entirety); and reverse transcriptase polymerase chain reaction (or RT-PCR, described in, for example, U.S. Pat. Nos. 5,322,770 and 5,310,652).
[0187] The PCR (or polymerase chain reaction) technique is well-known in the art and has been disclosed, for example, in Mullis and Faloona, Methods Enzymol., 155:350-355 (1987). In its simplest form, PCR is an in vitro method for the enzymatic synthesis of specific DNA sequences, using two primers that hybridize to opposite strands and flank the region of interest in the target DNA. A plurality of reaction cycles, each cycle comprising: a denaturation step, an annealing step, and a polymerization step, results in the exponential accumulation of a specific DNA fragment, see for example, “PCR Protocols: A Guide to Methods and Applications”, Innis (Ed.), 1990, Academic Press: New York; “PCR Strategies”, Innis (Ed.), 1995, Academic Press: New York; “Polymerase chain reaction: basic principles and automation in PCR: A Practical Approach”, McPherson et al. (Eds.), 1991, IRL Press: Oxford; Saiki et al., Nature 324:163-166 (1986). The termini of the amplified fragments are defined as the 5′ ends of the primers. Examples of DNA polymerases capable of producing amplification products in PCR reactions include, but are not limited to: E. coli DNA polymerase I, Klenow fragment of DNA polymerase I, T4 DNA polymerase, thermostable DNA polymerases isolated from Thermus aquaticus (Taq) which are available from a variety of sources (for example, Perkin Elmer), Thermus thermophilus (United States Biochemicals), Bacillus stereothermophilus (Bio-Rad), or Thermococcus litoralis (“Vent” polymerase, New England Biolabs). RNA target sequences may be amplified by reverse transcribing the mRNA into cDNA, and then performing PCR (RT-PCR), as described above. Alternatively, a single enzyme may be used for both steps as described in U.S. Pat. No. 5,322,770.
[0188] The duration and temperature of each step of a PCR cycle, as well as the number of cycles, are generally adjusted according to the stringency requirements in effect. Annealing temperature and timing are determined both by the efficiency with which a primer is expected to anneal to a template and the degree of mismatch that is to be tolerated. The ability to optimize the reaction cycle conditions is well within the knowledge of one of ordinary skill in the art. Although the number of reaction cycles may vary depending on the detection analysis being performed, it usually is at least 15, more usually at least 20, and may be as high as 60 or higher. However, in many situations, the number of reaction cycles typically ranges from about 20 to about 40.
[0189] The denaturation step of a PCR cycle generally comprises heating the reaction mixture to an elevated temperature and maintaining the mixture at the elevated temperature for a period of time sufficient for any double-stranded or hybridized nucleic acid present in the reaction mixture to dissociate. For denaturation, the temperature of the reaction mixture is usually raised to, and maintained at, a temperature ranging from about 85° C. to about 100° C., usually from about 90° C. to about 98° C., and more usually about 90° C. to about 94° C. for a period of time ranging from about 3 to about 120 seconds, usually from about 5 to about 30 seconds. In some embodiments, the first cycle is preceded by an elongated denaturation step ranging from about 1 to 10 minutes, usually from about 2 to 5 minutes.
[0190] Following denaturation, the reaction mixture is subjected to conditions sufficient for primer annealing to template DNA present in the mixture. The temperature to which the reaction mixture is lowered to achieve these conditions is usually chosen to provide optimal efficiency and specificity, and generally ranges from about 45° C. to about 75° C., usually from about 50° C. to about 70° C., and more usually from about 53° C. to about 55° C. Annealing conditions are generally maintained for a period of time ranging from about 15 seconds to about 30 minutes, usually from about 30 seconds to about 1 minute.
[0191] Following annealing of primer to template DNA or during annealing of primer to template DNA, the reaction mixture is subjected to conditions sufficient to provide for polymerization of nucleotides to the primer's end in a such manner that the primer is extended in a 5′ to 3′ direction using the DNA to which it is hybridized as a template (i.e., conditions sufficient for enzymatic production of primer extension product). To achieve primer extension conditions, the temperature of the reaction mixture is typically raised to a temperature ranging from about 65° C. to about 75° C., usually from about 67° C. to about 73° C., and maintained at that temperature for a period of time ranging from about 15 seconds to about 20 minutes, usually from about 30 seconds to about 5 minutes. In some embodiments, the final extension step is followed by an elongated extension step ranging from ranging from about 1 to 10 minutes, usually from about 2 to 5 minutes.
[0192] The above cycles of denaturation, annealing, and polymerization may be performed using an automated device typically known as a thermal cycler or thermocycler. Thermal cyclers that may be employed are described in U.S. Pat. Nos. 5,612,473; 5,602,756; 5,538,871; and 5,475,610. Thermal cyclers are commercially available, for example, from Perkin Elmer-Applied Biosystems (Norwalk, Conn.), BioRad (Hercules, Calif.), Roche Applied Science (Indianapolis, Ind.), and Stratagene (La Jolla, Calif.).
[0193] In some embodiments, one or both of the PCR reactions are “kinetic PCR” (kPCR) or “kinetic RT-PCR” (kRT-PCR), which are also referred to as “real-time PCR” and “real-time RT-PCR,” respectively. These methods involve detecting PCR products via a probe that provides a signal (typically a fluorescent signal) that is related to the amount of amplified product in the sample. Examples of commonly used probes used in kPCR and kRT-PCR include the following probes: TAQMAN® probes, Molecular Beacons probes, SCORPION® probes, and SYBR® Green probes. Briefly, TAQMAN® probes, Molecular Beacons, and SCORPION® probes each have a fluorescent reporter dye (also called a “fluor”) attached to the 5′ end of the probes and a quencher moiety coupled to the 3′ end of the probes. In the unhybridized state, the proximity of the fluor and the quench molecules prevents the detection of fluorescent signal from the probe. During PCR, when the polymerase replicates a template on which a probe is bound, the 5′-nuclease activity of the polymerase cleaves the probe thus, increasing fluorescence with each replication cycle. SYBR® Green probes binds double-stranded DNA and upon excitation emit light; thus as PCR product accumulates, fluorescence increases.
[0194] In some embodiments, the PCR reaction is used in a “single-plex” PCR assay. “Single-plex” refers to a single assay that is not carried out simultaneously with any other assays. Single-plex assays include individual assays that are carried out sequentially.
[0195] In some embodiments, the PCR reaction is used in a “multiplex” PCR assay. The term “multiplex” refers to multiple assays that are carried out simultaneously, in which detection and analysis steps are generally performed in parallel. Within the context of the present disclosure, a multiplex assay will include the use of the primers, alone or in combination with additional primers to identify, for example, an internal control, or an HCV virus variant along with one or more additional HCV variants or other viruses.
[0196] In some embodiments, a first amplification step amplifies a region of a target gene. In some embodiments the amplification product is less than about 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 250, 225, 200, 175, 150, 125, 100, or 75 nucleotides long.
Detection of Amplification Products
[0197] Amplification products generated using the oligonucleotides and methods of the present disclosure may be detected using a variety of methods known in the art.
[0198] In some embodiments, amplification products may simply be detected using agarose gel electrophoresis and visualization by ethidium bromide staining and exposure to ultraviolet (UV) light.
[0199] In some embodiments, the presence of a specific genotype can be shown by restriction enzyme analysis. For example, a specific nucleotide polymorphism can result in a nucleotide sequence comprising a restriction site which is absent from the nucleotide sequence of another allelic variant. Additionally or alternately, a specific nucleotide polymorphism can result in the elimination of a nucleotide sequence comprising a restriction site which is present in the nucleotide sequence of another allelic variant.
[0200] Examples of techniques for detecting differences of at least one nucleotide between two nucleic acids include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide probes may be prepared in which the known polymorphic nucleotide is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found, e.g., see Saiki et al., Nature 324:163 (1986); Saiki et al., Proc. Natl Acad. Sci USA 86:6230 (1989); and Wallace et al., Nucl. Acids Res. 6:3543 (1979). Such specific oligonucleotide hybridization techniques may be used for the simultaneous detection of several nucleotide changes in different polymorphic regions of DNA. For example, oligonucleotides having nucleotide sequences of specific allelic variants are attached to a hybridizing membrane and this membrane is then hybridized with labeled sample nucleic acid. Analysis of the hybridization signal will then reveal the identity of the nucleotides of the sample nucleic acid. Alternatively unlabeled sample nucleic acid may be immobilized and contacted with labeled oligonucleotides that hybridize selectively with specific allelic variants.
[0201] Real-time pyrophosphate DNA sequencing is yet another approach to detection of polymorphisms and polymorphic variants, e.g., see Alderborn et al., Genome Research, 10(8):1249-1258 (2000). Additional methods include, for example, PCR amplification in combination with denaturing high performance liquid chromatography (dHPLC), e.g., see Underhill et al., Genome Research, 7(10):996-1005 (1997).
[0202] In some embodiments, any of a variety of sequencing reactions known in the art can be used to directly sequence at least a portion of amplified DNA and detect allelic variants. The sequence can be compared with the sequences of known allelic variants to determine which one(s) are present in the sample. Exemplary sequencing reactions include those based on techniques developed by Maxam and Gilbert, Proc. Natl. Acad. Sci USA, 74:560 (1977) or Sanger, Proc. Nat. Acad. Sci 74:5463 (1977). It is also contemplated that any of a variety of automated sequencing procedures may be utilized when performing the subject assays, e.g., see Venter et al., Science, 291:1304-1351 (2001); Lander et al., Nature, 409:860-921 (2001), including sequencing by mass spectrometry, e.g., see U.S. Pat. No. 5,547,835 and PCT Patent Publication No. WO 94/16101 and WO 94/21822; U.S. Pat. No. 5,605,798 and PCT Patent Application No. PCT/US96/03651; Cohen et al., Adv. Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem. Biotechnol. 38:147-159 (1993). It will be evident to one skilled in the art that, for some embodiments, the occurrence of only one, two or three of the nucleic acid bases need be determined in the sequencing reaction. Yet other sequencing methods are disclosed, e.g., in U.S. Pat. Nos. 5,580,732; 5,571,676; 4,863,849; 5,302,509; PCT Patent Application Nos. WO 91/06678 and WO 93/21340; Canard et al., Gene 148:1-6 (1994); Metzker et al., Nucleic Acids Research 22:4259-4267 (1994) and U.S. Pat. Nos. 5,740,341 and 6,306,597.
[0203] In some embodiments, detection of an amplicon is performed using Real-time PCR. Real-time PCR has been developed to quantify amplified products during PCR reactions. Real-time PCR is based on the principles that emission of fluorescence from dyes directly or indirectly associated with the formation of newly-synthesized amplicons or the annealing of primers with DNA templates can be detected and is proportional to the amount of amplicons in each PCR cycle. Real-time PCR is carried out in a closed-tube format and is quantitative. Several methods are currently available for performing real-time PCR, such as utilizing TaqMan probes (U.S. Pat. Nos. 5,210,015 and 5,487,972, and Lee et al., Nucleic Acids Res. 21:3761-6, 1993), molecular beacons (U.S. Pat. Nos. 5,925,517 and 6,103,476, and Tyagi and Kramer, Nat. Biotechnol. 14:303-8, 1996), self-probing amplicons (scorpions) (U.S. Pat. No. 6,326,145, and Whitcombe et al., Nat. Biotechnol. 17:804-7, 1999), Amplisensor (Chen et al., Appl. Environ. Microbiol. 64:4210-6, 1998), Amplifluor (U.S. Pat. No. 6,117,635, and Nazarenko et al., Nucleic Acids Res. 25:2516-21, 1997, displacement hybridization probes (Li et al., Nucleic Acids Res. 30:E5, 2002), DzyNA-PCR (Todd et al., Clin. Chem. 46:625-30, 2000), fluorescent restriction enzyme detection (Cairns et al. Biochem. Biophys. Res. Commun. 318:684-90, 2004) and adjacent hybridization probes (U.S. Pat. No. 6,174,670 and Wittwer et al., Biotechniques 22:130-1, 134-8, 1997). Most of these probes consist of a pair of dyes (a reporter dye and an acceptor dye) that are involved in fluorescence resonance energy transfer (FRET), whereby the acceptor dye quenches the emission of the reporter dye. In general, the fluorescence-labeled probes increase the specificity of amplicon quantification.
[0204] In some embodiments, detection of an amplicon is performed using Real-time PCR coupled with a TaqMan assay. U.S. Pat. Nos. 5,210,015 and 5,487,972 describe the 5′ nuclease assay, also termed TaqMan assay. The TaqMan assay exploits the 5′ nuclease activity of Taq DNA Polymerase to cleave a TaqMan probe during PCR. The TaqMan probe contains a reporter dye at the 5′ end of the probe and a quencher dye at the 3′ end of the probe. During the reaction, cleavage of the probe separates the reporter dye and the quencher dye, resulting in increased fluorescence of the reporter. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye. When the probe is intact, the close proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence primarily by Förster-type energy transfer (Förster, 1948; Lakowicz, 1983). During PCR, if the target of interest is present, the probe specifically anneals between the forward and reverse primer sites. The 5′ to 3′ nucleolytic activity of the Taq DNA polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target. The probe fragments are then displaced from the target, and polymerization of the strand continues. The 3′ end of the probe is blocked to prevent extension of the probe during PCR. This process occurs in every cycle and does not interfere with the exponential accumulation of the product.
Compositions and Kits
[0205] In some embodiments, the present disclosure provides kits comprising materials useful for the amplification and detection or sequencing of specific AAV constructs according to methods described herein. The inventive kits may be used by diagnostic laboratories, experimental laboratories, or practitioners.
[0206] Materials and reagents useful for the detection or sequencing of specific AAV constructs according to the present disclosure may be assembled together in a kit. In some embodiments, an inventive kit comprises at least one inventive primer set, and optionally, reverse transcription and/or amplification reaction reagents. In some embodiments, a kit comprises reagents which render the procedure specific. Thus, a kit intended to be used for the detection of a particular specific AAV construct variant preferably comprises primer sets described herein that can be used to amplify a particular specific AAV construct target sequence of interest. A kit intended to be used for the multiplex detection of a plurality of specific AAV construct target sequences and/or other viruses preferably comprises a plurality of primer sets (optionally in separate containers) described herein that can be used to amplify specific AAV construct target sequences described herein.
[0207] Suitable reverse transcription/amplification reaction reagents that can be included in an inventive kit include, for example, one or more of: buffers; enzymes having reverse transcriptase and/or polymerase activity; enzyme cofactors such as magnesium or manganese; salts; nicotinamide adenide dinuclease (NAD); and deoxynucleoside triphosphates (dNTPs) such as, for example, deoxyadenosine triphospate; deoxyguanosine triphosphate, deoxycytidine triphosphate and deoxythymidine triphosphate, biotinylated dNTPs, suitable for carrying out the amplification reactions.
[0208] Depending on the procedure, the kit may further comprise one or more of: wash buffers and/or reagents, hybridization buffers and/or reagents, labeling buffers and/or reagents, and detection means. The buffers and/or reagents included in a kit are preferably optimized for the particular amplification/detection technique for which the kit is intended. Protocols for using these buffers and reagents for performing different steps of the procedure may also be included in the kit.
[0209] Furthermore, the kits may be provided with an internal control as a check on the amplification procedure and to prevent occurrence of false negative test results due to failures in the amplification procedure. An optimal control sequence is selected in such a way that it will not compete with the target nucleic acid sequence in the amplification reaction (as described above).
[0210] Kits may also contain reagents for the isolation of nucleic acids from biological specimen prior to amplification and/or for the purification or separation of AAV particles before nucleic acid extraction.
[0211] The reagents may be supplied in a solid (e.g., lyophilized) or liquid form. The kits of the present disclosure optionally comprise different containers (e.g., vial, ampoule, test tube, flask or bottle) for each individual buffer and/or reagent. Each component will generally be suitable as aliquoted in its respective container or provided in a concentrated form. Other containers suitable for conducting certain steps of the amplification/detection assay may also be provided. The individual containers of the kit are preferably maintained in close confinement for commercial sale.
[0212] The kit may also comprise instructions for using the amplification reaction reagents and primer sets or primer/probe sets according to the present disclosure. Instructions for using the kit according to one or more methods of the present disclosure may comprise instructions for processing the biological sample, extracting nucleic acid molecules, and/or performing the test; instructions for interpreting the results as well as a notice in the form prescribed by a governmental agency (e.g., FDA) regulating the manufacture, use or sale of pharmaceuticals or biological products.
[0213] The disclosure is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the disclosure should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations that become evident as a result of the teaching provided herein.
[0214] For example, other assays, including those described in the Example section herein as well as those that are known in the art, can also be used in accordance with the present disclosure.
EXAMPLES
Reagents, Equipment, Methods, and Consumables Utilized For Immediate Examples—Reagents
[0215]
TABLE-US-00004 Storage Reagent Source Catalog # Condition MNase (2000 U/μL) New M0247S −10° C. to England −30° C. Biosciences (NEB) 0.5M EDTA, pH 8.0 ThermoFisher 15575020 RT Nuclease-free water Ambion AM9937 RT 100X BSA NEB B9001S −10° C. to −30° C. 10X MNase buffer NEB B0247S −10° C. to −30° C. Pre-treatment buffer In-house BKY-BPR-0003 RT Molecular Grade, MLS RT Nuclease Free Water PerfeCTa Toughmix, QuantaBio 95140-050K −10° C. to UNG, low ROX −30° C. 20X Hem-B Oligo Mix Siemens BKY-BPR-0002 −10° C. to −30° C. VERSANT ® kPCR Siemens Box 1 10286026 RT Sample Preparation Healthcare Box 2 10286027 2 to 8° C. 1.0 Reagents Diagnostics Inc Siemens Sample In-house NA 2° C. to 8° C. Diluent 1 (SSD1) Siemens Storage In-house NA 2° C. to 8° C. Buffer 2 (SSB2) Siemens Pretreatment In-house NA RT Buffer (SPB)
Equipment and Consumables
[0216]
TABLE-US-00005 Description Vendor Part # Single-channel adjustable pipettes MLS N/A (L10, L20, L200, and L1000) Freezer (−10° C. to −30° C.) MLS NA Bleach, unscented (0.5% sodium MLS NA hypochlorite) Incubator MLS NA LTS 20 μL Filter Tips Rainin 17014961 LTS 200 μL Filter Tips Rainin 17014963 LTS 1000 μL Filter Tips Rainin 17014967 3.5 mL Sarstedt Tubes with VWR 101093-666 enclosed neutral cap Microcentrifuge tubes MLS NA (0.5, 1.5, or 2.0 mL) Amber Microcentrifuge tubes (2.0 mL) MLS NA MicroAmp ® Optical 96-well Applied 4346907 Fast reaction plate, 0.1 mL, MicroAmp ® Optical Adhesive Film, Applied 4311971 100 each Dedicated single-channel adjustable MLS NA pipettes (P2, P10, P20, P200, and P1000) Multi-channel pipette (P10, P20) MLS NA Personal Protective Equipment (PPE) MLS NA Dead-air hood with UV light MLS NA Microcentrifuge MLS NA Vortex Mixer MLS NA Allegra 25R Centrifuge, 60 Hz, 208 V Beckman Coulter QuantStudio ™ 7 Flex Real-Time or DX Thermo PCR System with 96-Well Fast Block Fisher S5700 Swinging Bucket Rotor for Beckman 369434, 368954 96-well plate with microplate carrier Coulter Refrigerator (2° C. to 8° C.) MLS NA Sub-Freezer (−60° C. to −90° C.) MLS NA VERSANT ® kPCR Molecular SHDI NA System Sample Preparation Module (SP Module) Biosafety Cabinet MLS NA SP1.0 Reagent Trough SHDI SMN 10489008 (200 mL & 50 mL) 1000-μL pipette tips SHDI SMN 10282929 300-μL pipette tips SHDI SMN 10282930 96-well, 2-mL nuclease free, SHDI SMN 10283255 sterile deep well plates Barcoded 96-well semi-skirted SHDI SMN 10282998 polypropylene plates for kPCR PCR Adapter Plate SHDI 96067-01 Blue Absorbent Pads (bench protectors) MLS NA 70% ethanol MLS NA Bleach, unscented MLS NA Microcide SQ SHDI SMN 10361387 Deionized water In-house Hamilton Tip Disposal Bags (200 ea.) SHDI SMN 10282938
EXAMPLE 1: General Methodologies
[0217] The present example provides an overview of a number of generalizable assays and/or treatments utilized during sample preparation and quantification.
[0218] MNase Treatment—MNase Treatment procedure was performed prior to sample extraction and in line with manufacturer recommendations.
[0219] Quantification by qPCR for the AAV Shedding Assays—Extracted DNA was tested in a qPCR reaction using 54, of DNA extract. The concentration of AAV viral DNA in samples was determined by plotting their Ct values against Ct values obtained from extracted AAV viral calibrators in buffer at known concentrations. QuantStudio™ 7 Flex or Dx Real-Time PCR Systems were utilized for qPCR analysis. The concentration of AAV viral DNA in plasma, urine, semen and saliva samples were reported as viral genomes (vg)/mL and in PBMC samples reported as viral genomes (vg)/1 μg genomic DNA.
[0220] Sample Preparation for AAV Shedding Assay—Samples were prepared utilizing viral DNA extraction and purification processes according to the VERSANT® kPCR Molecular System—Stand Alone Sample Preparation module (SASP). Viral DNA samples were separated and purified for use in amplification procedures according to manufacturer instructions.
EXAMPLE 2: Identifying and Optimizing Primers for Duchenne Muscular Dystrophy AAV Gene Therapy Shedding Quantification
[0221] The present example demonstrates the utility, accuracy, sensitivity, and precision of assays performed utilizing oligonucleotide sequences described herein. This shedding assay detects and quantifies an exemplary viral DNA (e.g., exemplary DMD AAV construct) extracted from urine, whole blood, stool and saliva. Exemplary DMD AAV construct is an adeno-associated virus encoding mini-dystrophin.
[0222] Extracted DNA was tested in a qPCR reaction using 5 μL of DNA extract. A linear regression curve was generated by plotting Ct values of extracted exemplary DMD AAV construct viral calibrators in buffer against their known concentrations (log transformed, see
[0223] Present FDA guidance requires qPCR to be able to detect<50 copies of vector/1 μg genomic DNA with 95% confidence. The qPCR quantitation limit was determined using a panel of linearized plasmid containing the exemplary DMD AAV construct viral genome spiked into human genomic DNA. N=444 was chosen for 25 copies of vector/1 μg genomic DNA to ensure that a 95% detection rate with enough confidence could be achieved (summarized below).
Primary Screening of Exemplary Primers and/or Probes
[0224] Primers were designed targeting select regions of an exemplary DMD AAV construct (see
[0225] Certain primer and probe combinations were determined to work well under tested assay conditions. As shown in the figures and further herein, certain primer and probe combinations provided a desirable profile. However, alternative primer and/or probe combinations can be screened as shown to determine desirable profiles for other conditions, samples, etc., and the present disclosure recognizes that multiple primer and/or probe combinations may be suitable for accurate and reliable detection and/or quantification. The data also confirms that select primer and probe combinations work well when multiplexed, for example, with internal control primer and probe combinations. In some embodiments, those of skill in the art will understand that certain characteristics may be more or less desirable and that selection of a set of primers and probes for a particular application may be dependent on the weighing of certain characteristics such as multiplexing capacity, maximum fluorescence, slope, minimum CT, Tm, oligonucleotide cross reactivity, specificity for DMD AAV constructs in the presence of background genomic DNA, or any combination of all or any of these factors. Nonetheless, the data provided herein established that the disclosed primers and/or probes worked well for the detection of AAV constructs in biological samples. At least one or more forward primers, at least one or more reverse primers, and at least one or more probes were found suitable for one or more primary screening criteria (see e.g., Table 1, Table 2, and
TABLE-US-00006 TABLE 1 Primary Screening of Certain Oligonucleotide Combinations targeting alternative Exemplary DMD AAV Construct regions. Exem- plary DMD AAV Con- Amplicon struct Size Assay Region Fwd-P Rev-P Detection [bp] 1 Region DMD- DMD- DMD- 98 1 FWD-A REV-B PROBE-A (SEQ ID (SEQ ID (SEQ ID NO: 1) NO: 11) NO: 17) 2 DMD- DMD- DMD- 95 FWD-B REV-A PROBE-A (SEQ ID (SEQ ID (SEQ ID NO: 3) NO: 9) NO: 17) 3 Region DMD- DMD- DMD- 108 2 FWD-E REV-E PROBE-F (SEQ ID (SEQ ID (SEQ ID NO: 63) NO: 67) NO: 73) 4 DMD- DMD- DMD- 78 FWD-E REV-F PROBE-F (SEQ ID (SEQ ID (SEQ ID NO: 63) NO: 69) NO: 73) 5 Region DMD- DMD- DMD- 70 3 FWD-F REV-G PROBE-G (SEQ ID (SEQ ID (SEQ ID NO: 65) NO: 71) NO: 77) Ampl. effi- 62500 15625 3906.25 976.56 cien- Assay copies copies copies copies slope cy 1 19.50 21.48 23.48 25.77 −3.45 94.8 2 19.62 21.55 23.77 25.73 −3.41 96.3 3 19.31 21.38 23.35 25.32 −3.32 99.9 4 19.36 21.38 23.41 25.57 −3.43 95.7 5 16.97 19.27 21.10 23.51 −3.56 90.90
TABLE-US-00007 TABLE 2 Primary Screening of Certain Multiplex Assays for Exemplary DMD AAV Constructs Amplicon As- DNA Detec- Size say Source Fwd-P Rev-P tion [bp] 1 Exem- DMD- DMD- DMD- 70 plary FWD-F REV-G PROBE-G DMD (SEQ ID (SEQ ID (SEQ ID AAV NO: 65) NO: 71) NO: 77) Con- struct 2 IC IC-F IC-R IC-P — As- 62500 15625 3906.25 976.56 488.28 Ampl. say copies copies copies copies copies slope efficiency 1 18.72 20.85 23.47 25.01 27.02 −3.57 90.66 2 17.56 19.48 21.84 23.88 25.37 −3.54 91.72
Data Calculations and Reporting
[0226] The observed data were fitted with a nonlinear regression model as follows:
Precision=β.sub.0+β.sub.1×e.sup.(β.sup.
[0227] where β.sub.0, β.sub.1, and β.sub.2 are the coefficients of the model, and c is the random error term. JMP (Version 14.1, SAS Institute) was utilized for calculations with an exponential 3P fit curve model.
[0228] The Lower Limit of Quantitation (LLoQ) using the precision profile method was calculated as the concentration at the upper 95% confidence limit of the fitted curve at 20% CV.
[0229] The lower limit of quantification (LLoQ) for qPCR was determined to be 50 copies of viral genome/1 μg genomic DNA based on the criteria of≥95% detection, ≤20% variability and within 0.125 LOG of difference from the target value, exceeding the FDA guidance requirements.
[0230] The shedding assay detection range is summarized below. If the titer is detected to be above the upper quantitation limit, the sample was reported as ALQ (above limit of quantitation); if the titer is determined to be below the lower quantitation limit, the sample was reported as BLQ (below limit of quantitation); and if the titer is within the quantitation range, a numeric concentration was reported.
[0231] The raw signals for exemplary DMD AAV construct whole blood, saliva, stool and urine were determined using QuantStudio™ 7 Flex and QuantStudio™ Software v1.1. The concentrations of exemplary DMD AAV construct were calculated using a linear regression curve, which is generated by plotting Ct values of extracted exemplary DMD AAV construct viral calibrators in buffer against their known concentrations (log transformed). The concentration of exemplary DMD AAV construct viral DNA in samples is determined by fitting the Ct values in this model. This was done in Microsoft Excel, Microsoft Office 363 Pro version 1909. For tables, final concentrations were reported in viral genome (vg)/mL, except for stool where it was reported in vg/mg, to at least three significant figures. Precision (% CV) was reported to the nearest 0.1%.
[0232] Statistical Analysis
[0233] Accuracy—Titer data were considered log-normally distributed and analyzed following log 10 transformation. exemplary DMD AAV construct shedding assay accuracy was determined using the same panels described in the analytical sensitivity section below. Accuracy was calculated by the difference between observed value and the expected value after log transformation, and the target requirement was within ±0.5 log bias of expected value across the quantitative range. exemplary DMD AAV construct shedding assay linearity and accuracy for each sample type is summarized below.
[0234] Linearity—The assay linearity was assessed by fitting a linear regression model as follows
Log 10(Quantitation)=β0+β1×Log 10(Input Concentration)+ε
[0235] Where β0 is intercept, β1 is slope and c is the random error term. The coefficient of determination R.sup.2 was calculated based on this model. Linearity is established if R.sup.2 is above 0.95. Accuracy and linearity analysis was performed using R (Version 3.5.1 2018-07-02, https://cran.rproject.org/).
[0236] Precision—Assay within-laboratory reproducibility, or precision, was estimated using the following variance components model for each concentration level,
Quantitation=intercept+instrument+run+within-run (error)
[0237] Where instrument, run, and within-run errors were treated as random effects. Variance components were estimated via a random effect model. The total variance, δ.sup.2, which accounted for all between-instrument, between-run, and within-run effects, was estimated as the sum of the individual variance components. The measure of reproducibility is the percent coefficient of variation (% CV), or the total variance divided by the mean. Variance components were analyzed in JMP (Version 13.1, SAS Institute) using REML (Restricted Maximum Likelihood) model (https://www.jmp.com/support/help/14-2/restricted-maximumlikelihood-reml-model.shtml).
[0238] Specificity—Assay specificity was calculated as the ratio of identified negative samples to all unspiked (true negative) samples. The assay specificity for exemplary DMD AAV construct shedding was determined with 20-24 biological replicates (20 for saliva, stool, and urine, and 24 for whole blood), using normal human saliva, stool, urine and whole blood samples without any viral spike. The specificity was higher than 95% in all sample type tested.
[0239] Analytical Sensitivity—For measurement of analytical sensitivity, the observed data were fitted with a nonlinear regression model as follows:
Precision=β.sub.0+β.sub.1×e{circumflex over ( )}((β.sub.2×log 10(Observed Concentration)))+ε
[0240] where β0, β1, and β2 are the coefficients of the model, and c is the random error term. EVP (Version 14.1, SAS Institute) was utilized for calculations with an exponential 3P fit curve model.
[0241] The Lower Limit of Quantitation (LLoQ) using the precision profile method was calculated as the concentration at the upper 95% confidence limit of the fitted curve at 30% CV.
[0242] Analytical sensitivity was determined in a two-step process. In Step 1, for Saliva, Stool and Urine, an up to 12-replicate panel was tested on two different VERSANT kPCR extraction systems to estimate the Lower Limit of Quantitation (LLoQ), for Blood only one VERSANT kPCR extraction system was used to test at least 6 replicates each. In Step 2, up to 36 biological replicates near the estimated LLoQ level were utilized to further refine LLoQ for all sample types. The concentration at the upper 95% confidence limit of the fitted curve at 30% CV and within ±0.5 LOG of bias was determined as the LLoQ for that sample type. Exemplary DMD AAV construct shedding assay analytical sensitivity for each sample type are summarized below.
[0243] RESULTS
[0244] Shedding Assay Performance Summary—The shedding assay provided a quantitative result for saliva, stool, urine, and whole blood samples within corresponding assay ranges. The shedding assay detection range is summarized in Table 3. If the titer was detected to be above the upper quantitation limit, the sample was reported as AQL (above quantitation limit); if the titer was determined to be below the lower quantitation limit, the sample was be reported as BQL (below quantitation limit); and if the titer was within the quantitation range, a numeric concentration was reported.
TABLE-US-00008 TABLE 3 Performance Evaluation Summary Lower Limit of Upper Limit of Matrix Quantification (LLOQ) Quantification (ULoQ) Saliva 3.67E+03 vg/mL 9.84E+08 vg/mL Stool 3.90E+01 vg/mg 1.28E+07 vg/mg Urine 4.26E+03 vg/mL 1.14E+09 vg/mL Whole Blood 8.45E+03 vg/mL 7.63E+08 vg/mL
[0245] DNase Treatment Performance Summary—MNase treatment was chosen as nuclease for DNase treatment assessment. MNase is a DNA and RNA endonuclease and is able to cleave double-stranded DNA (dsDNA), single-stranded DNA (ssDNA) and RNA. MNase has higher activity than DNase I (MNase at 2,000,000 units/mL and DNase I at 2,000 units/mL are commercially available through New England Biolabs). Incomplete digestion of unprotected DNA was observed with DNase I (up to 250 units, limited by the stock concentration) in reaction buffer system. In comparison, treatment with 4,000 units of MNase eliminated the unprotected deoxynucleic acid (both dsDNA and ssDNA) and did not decrease exemplary Hem-B AAV construct recovery from intact viral particles, in all sample types tested (see immediate example 3).
[0246] MNase treatment on intact exemplary DMD AAV construct viral DNA recovery was assessed in all sample types, except whole blood, at a concentration near the LLoQ, with a minimum of 12 replicates. No significant difference was observed between MNase treated or untreated samples for all sample types (see Table 4 below and
TABLE-US-00009 TABLE 4 MNase treatment summary Untreated MNase treated Log Matrix (vg/mL) N = 36 (vg/mL) N = 12 Difference % Loss Saliva 3.45E+03 2.92E+03 −0.073 15.39% Urine 4.85E+03 5.15E+03 0.026 −6.10% Stool 4.06E+03 3.41E+03 −0.076 16.05% Linearize 1.06E+05 0 NA 100% plasmid
[0247] qPCR Performance—The FDA guidance requires qPCR to be able to detect<50 copies of vector/1 μg genomic DNA with 95% confidence. The qPCR quantitation limit was determined using a panel of linearized plasmid containing the exemplary DMD AAV construct viral genome spiked into human genomic DNA. N=444 was chosen for 25 copies of vector/1 μg genomic DNA to ensure that a 95% detection rate with enough confidence can be achieved (summarized in
TABLE-US-00010 TABLE 5 Analytical Sensitivity Results Summary (LloQ) Accuracy LloQ (LogObs- [95% Expected Observed No. LogExp, CI] (vg/1 μg (vg/1 μg of No. Detection Within ± Precision (vg/1 μg gDNA) gDNA) Reps Detected Rate 0.125LOG) (<20% CV) gDNA) 100 99.7 12 12 100.0% −0.0013 11.93% 50 75 39.0 12 12 100.0% −0.0059 16.08% 50 52.2 16 16 100.0% 0.0187 17.17% 37 39.0 12 12 100.0% 0.0467 20.52% 25 23.8 12 12 100.0% −0.0208 32.20% 20 22.0 12 12 100.0% 0.0406 29.59% 10 10.7 12 12 100.0% 0.0291 42.67%
TABLE-US-00011 TABLE 6 qPCR Limit of Detection Expected Observed No. of No. No. of Detection (vg/1 μg gDNA) (vg/1 μg gDNA) Reps Detected Runs Rate 25 27.2 444 444 3 100.0%
[0248] Accuracy—Titer data were considered to be log-normally distributed and were analyzed following log 10 transformation. Exemplary DMD AAV construct shedding assay accuracy was determined using the same sample panels described above for analytical sensitivity. Accuracy was calculated by the difference between observed value and the expected value after log transformation, and the target requirement was with ±0.5 log bias of expected value across the quantitative range. Exemplary DMD AAV construct shedding assay linearity and accuracy for each sample type was summarized and is presented in Tables 7-10.
TABLE-US-00012 TABLE 7 Assessment of Accuracy of Shedding Assay of Exemplary DMD AAV Construct in Saliva Observed Expected Concentration LogQTY LogQTY No. of Log Difference (vg/mL) (Log vg/mL) (Log vg/mL) Reps (LogObs-LogExp) 9.8E+08 8.99 8.99 12 0.01 9.6E+06 6.97 6.98 12 0.01 1.0E+05 4.96 5.00 12 0.04 1.2E+04 4.12 4.07 10 −0.06 9.2E+03 3.95 3.96 12 0.01 6.5E+03 3.78 3.81 36 0.04 6.3E+03 3.79 3.80 12 0.00 5.0E+03 3.73 3.70 12 −0.03 3.8E+03 3.55 3.58 12 0.03 3.4E+03 3.53 3.54 36 0.01 1.8E+03 3.27 3.24 36 −0.03
TABLE-US-00013 TABLE 8 Assessment of Accuracy of Shedding Assay of Exemplary DMD AAV Construct in Stool* Observed Expected Concentration LogQTY LogQTY No. of Log Difference (vg/mg) (Log vg/mg) (Log vg/mg) Reps (LogObs-LogExp) 1.28E+07 6.99 7.11 11 0.12 1.40E+05 4.98 5.15 12 0.17 1.35E+03 2.97 3.13 12 0.16 1.29E+02 1.97 2.11 10 0.14 9.86E+01 1.95 1.99 35 0.05 6.70E+01 1.67 1.83 12 0.16 4.91E+01 1.62 1.69 36 0.07 4.25E+01 1.57 1.63 11 0.06 4.06E+01 1.53 1.61 36 0.08 3.63E+01 1.51 1.56 35 0.05 3.07E+01 1.37 1.49 12 0.12 2.08E+01 1.15 1.32 12 0.17 *Stool was suspended in 10 volume (w/v) of 1X PBS before extraction.
TABLE-US-00014 TABLE 9 Assessment of Accuracy of Shedding Assay of Exemplary DMD AAV Construct in Urine Observed Expected Concentration LogQTY LogQTY No. of Log Difference (vg/mL) (Log vg/mL) (Log vg/mL) Reps (LogObs-LogExp) 1.1E+09 9.00 9.06 12 0.06 1.2E+07 6.98 7.07 12 0.08 1.1E+05 4.98 5.05 12 0.07 1.8E+04 4.15 4.26 12 0.11 1.2E+04 3.97 4.07 12 0.10 8.7E+03 3.88 3.94 12 0.06 6.6E+03 3.75 3.82 12 0.07 4.9E+03 3.67 3.69 36 0.01 4.1E+03 3.58 3.61 11 0.03 3.9E+03 3.63 3.59 36 −0.03 3.3E+03 3.55 3.51 36 −0.04
TABLE-US-00015 TABLE 10 Assessment of Accuracy of Shedding Assay of Exemplary DMD AAV Construct in Whole Blood Observed Expected Concentration LogQTY LogQTY No. of Log Difference (vg/mL) (Log vg/mL) (Log vg/mL) Reps (LogObs-LogExp) 7.63E+08 9.00 8.88 6 −0.12 7.80E+06 7.01 6.89 8 −0.11 1.25E+05 5.03 5.10 36 0.07 8.95E+04 5.00 4.95 9 −0.05 1.67E+04 4.21 4.22 36 0.02 1.32E+04 4.19 4.12 9 −0.07 9.93E+03 4.03 4.00 35 −0.04 7.89E+03 4.01 3.90 9 −0.12 6.70E+03 3.92 3.83 9 −0.09 6.47E+03 3.94 3.81 35 −0.12 3.68E+03 3.79 3.57 8 −0.23 2.26E+03 3.62 3.35 8 −0.26
[0249] Specificity—Assay specificity was calculated as the ratio of identified negative samples to all unspiked (true negative) samples. The assay specificity for exemplary DMD AAV construct shedding was determined with 20-24 biological replicates (20 for saliva, stool, and urine, and 24 for whole blood), using normal human saliva, stool, urine and whole blood samples without any viral spike. The specificity was higher than 95% in all sample type tested.
[0250] Analytical Sensitivity—Analytical sensitivity was determined in a two-step process. In Step 1, for Saliva, Stool and Urine, an up to 12-replicate panel was tested on two different VERSANT kPCR extraction systems to estimate the Lower Limit of Quantitation (LLoQ), for Blood only one VERSANT kPCR extraction system was used to test at least 6 replicates each. In Step 2, up to 36 biological replicates near the estimated LLoQ level were utilized to further refine LLoQ for all sample types. The concentration at the upper 95% confidence limit of the fitted curve at 30% CV and within ±0.5 LOG of bias was determined as the LLoQ for that sample type. Exemplary DMD AAV construct shedding assay analytical sensitivity for each sample type are summarized below in Tables 11-14.
TABLE-US-00016 TABLE 11 Analytical Sensitivity and Specificity for Exemplary DMD AAV Construct in Human Saliva Concen- No. LloQ tration of No. Detection (LogObs- Total [95% CI] (vg/mL) Reps Detected Rate LogExp) % CV (vg/mL) 9.84E+08 12 12 100.0% 0.006 10.61% 3670 9.58E+06 12 12 100.0% 0.012 7.67% 9.98E+04 12 12 100.0% 0.040 12.11% 1.17E+04 12 10 100.0% −0.059 17.61% 9.17E+03 12 12 100.0% 0.014 16.78% 6.53E+03 36 36 100.0% 0.036 16.84% 6.27E+03 12 12 100.0% 0.002 26.80% 5.01E+03 12 12 100.0% −0.026 18.81% 3.81E+03 12 12 100.0% 0.032 29.93% 3.44E+03 36 36 100.0% 0.008 17.50% 1.75E+03 36 36 100.0% −0.030 30.19%
TABLE-US-00017 TABLE 12 Analytical Sensitivity and Specificity for Exemplary DMD AAV Construct in Human Stool* Concen- No. LloQ tration of No. Detection (LogObs- Total [95% CI] (vg/mg) Reps Detected Rate LogExp) % CV (vg/mg) 1.28E+07 11 11 100.0% 0.122 19.52% 39 1.40E+05 12 12 100.0% 0.168 16.55% 1.35E+03 12 12 100.0% 0.158 9.47% 1.29E+02 12 10 100.0% 0.139 16.48% 9.86E+01 36 35 100.0% 0.047 20.16% 6.70E+01 12 12 100.0% 0.158 18.56% 4.91E+01 36 36 100.0% 0.069 19.30% 4.25E+01 12 11 100.0% 0.057 25.61% 4.06E+01 36 36 100.0% 0.081 21.48% 3.63E+01 36 35 100.0% 0.045 21.99% 3.07E+01 12 12 100.0% 0.118 43.82% 2.08E+01 12 12 100.0% 0.172 39.11% *Stool was suspended in 10 volume (w/v) of 1X PBS before extraction.
TABLE-US-00018 TABLE 13 Analytical Sensitivity and Specificity for Exemplary DMD AAV Construct in Human Urine Concen- No. LloQ tration of No. Detection (LogObs- Total [95% CI] (vg/mL) Reps Detected Rate LogExp) % CV (vg/mL) 1.14E+09 12 12 100.0% 0.061 16.02% 4263 1.17E+07 12 12 100.0% 0.083 17.90% 1.12E+05 12 12 100.0% 0.070 14.03% 1.83E+04 12 12 100.0% 0.110 13.88% 1.17E+04 12 12 100.0% 0.095 17.88% 8.67E+03 12 12 100.0% 0.061 19.96% 6.62E+03 12 12 100.0% 0.069 14.13% 4.85E+03 36 36 100.0% 0.013 20.85% 4.05E+03 12 11 100.0% 0.030 33.65% 3.92E+03 36 36 100.0% −0.034 25.13% 3.25E+03 36 36 100.0% −0.036 26.56%
TABLE-US-00019 TABLE 14 Analytical Sensitivity and Specificity for Exemplary DMD AAV Construct in Human Whole Blood Concen- No. LloQ tration of No. Detection (LogObs- Total [95% CI] (vg/mL) Reps Detected Rate LogExp) % CV (vg/mL) 7.63E+08 6 6 100.0% −0.121 14.94% 8451 7.80E+06 9 8 100.0% −0.114 21.68% 1.25E+05 36 36 100.0% 0.074 20.01% 8.95E+04 9 9 100.0% −0.051 22.74% 1.67E+04 36 36 100.0% 0.015 20.81% 1.32E+04 9 9 100.0% −0.066 18.70% 9.93E+03 36 35 100.0% −0.037 28.45% 7.89E+03 9 9 100.0% −0.117 22.61% 6.70E+03 9 9 100.0% −0.090 34.12% 6.47E+03 36 35 100.0% −0.124 28.98% 3.68E+03 9 8 100.0% −0.225 40.85% 2.26E+03 9 8 100.0% −0.264 43.47%
[0251] Ambient temperature, Repeated Freeze/Thaw, and Long-term −80° C. Storage Stability of PBMCs, Saliva, Urine and Stool—Stability of exemplary DMD AAV construct virus in PBMCs, saliva, stool and urine stored at ambient temperature, long term storage at −80° C. for up to 6 months, and freeze/thaw cycling was assessed. Pooled samples were spiked at 1.0E+05 vg/mL (post 5E+6 cells/mL suspension of PBMCs and 1:10 (w:v) suspension of stool), and three biological replicates per condition were tested at indicated time points. Exemplary DMD AAV construct viral genome titer was within ±0.5 LOG of the titer determined at time 0 for up to 6 months. Long-term −80° C. Storage Stability results are summary in
TABLE-US-00020 TABLE 15 Long Term Stability of Exemplary DMD AAV Construct Stored and Frozen at −80° C. Log Mean Log Mean Timepoint No. of Titer [log Diff from CV Recovery Matrix (months) Replicates vg/mL] T0 [%] [%] Saliva 0 3 6.20 NA 1.43 NA 1 3 6.02 −0.12 3.77 75.90 2 3 6.17 −0.03 3.62 93.44 3 3 6.10 −0.10 1.64 79.46 6 3 6.20 0.01 9.99 101.39 9 3 6.03 −0.17 3.36 68.29 Stool 0 3 5.85 NA 5.66 NA 1 3 5.86 0.01 38.87 103.46 2 3 5.81 −0.04 9.64 91.11 3 3 5.81 −0.04 9.89 91.09 6 3 5.79 −0.06 13.63 87.68 9 3 5.72 −0.13 6.12 73.84 Urine 0 3 6.24 NA 0.80 NA 1 3 6.20 −0.03 0.29 92.95 2 3 6.26 0.02 1.11 105.56 3 3 6.16 −0.07 3.53 84.58 6 3 6.19 −0.05 3.50 89.03 9 3 6.18 −0.05 2.45 88.11 Whole 0 3 5.72 NA 3.68 NA Blood 1 3 5.72 0.00 11.22 100.05 2 3 5.77 0.05 4.61 112.44 3 3 5.73 0.01 13.01 103.47 6 3 5.58 −0.13 16.61 73.59 9 3 5.76 0.05 7.55 111.52
EXAMPLE 3: Identifying and Optimizing Primers for Exemplary Hemophilia B AAV Gene Therapy Shedding Quantification
[0252] The present example demonstrates the utility, accuracy, sensitivity, and precision of assays performed utilizing oligonucleotide sequences described herein. This shedding assay detects and quantifies an exemplary viral DNA (e.g., exemplary Hem-B AAV construct) extracted from urine, plasma, semen, whole blood, stool and saliva. Exemplary Hem-B AAV construct is an adeno-associated virus encoding factor IX. Extracted DNA was tested in a qPCR reaction using 5 μL of DNA extract. A linear regression curve was generated by plotting Ct values of extracted exemplary Hem-B AAV construct viral calibrators in buffer against their known concentrations (log transformed, see
[0253] The FDA guidance requires qPCR to be able to detect<50 copies of vector/1 μg genomic DNA with 95% confidence. The qPCR quantitation limit was determined using a panel of linearized plasmid containing the exemplary Hem-B AAV construct viral genome spiked into human genomic DNA. Linearized plasmid at different concentrations was tested with different numbers of replicates to assess the quantification limits. N=75 was chosen for the lowest concentration to ensure a 95% detection rate with enough confidence. The lower limit of quantification (LLoQ) for qPCR was determined to be 31 copies of viral genome/1 μg genomic DNA based on the criteria of ≥95% detection, <20% variability and within 0.125 LOG of difference from the target value (summarized below).
[0254] The present example demonstrates the utility, accuracy, sensitivity, and precision of assays performed utilizing oligonucleotide sequences described herein.
[0255] Primary Screening of Exemplary Primers and/or Probes
[0256] Primers were designed targeting select regions of an exemplary HemB AAV construct (see
[0257] Certain primer and probe combinations were determined to work well under tested assay conditions. As shown in the figures and further herein, certain primer and probe combinations provided a desirable profile. However, alternative primer and/or probe combinations can be screened as shown to determine desirable profiles for other conditions, samples, etc., and the present disclosure recognizes that multiple primer and/or probe combinations may be suitable for accurate and reliable detection and/or quantification. The data also confirms that select primer and probe combinations work well when multiplexed, for example, with internal control primer and probe combinations. In some embodiments, those of skill in the art will understand that certain characteristics may be more or less desirable and that selection of a set of primers and probes for a particular application may be dependent on the weighing of certain characteristics such as multiplexing capacity, maximum fluorescence, slope, minimum CT, Tm, oligonucleotide cross reactivity, specificity for HemB AAV constructs in the presence of background genomic DNA, or any combination of all or any of these factors. Nonetheless, the data provided herein established that the disclosed primers and/or probes worked well for the detection of AAV constructs in biological samples. At least one or more forward primers, at least one or more reverse primers, and at least one or more probes were found suitable for one or more primary screening criteria (for certain results see e.g., Table 16-18, and
TABLE-US-00021 TABLE 16 Primary Screening of Certain Oligonucleotide Combinations targeting alternative Exemplary HEM-B AAV Construct regions. Exem- plary Hem- BAAV Con- Amplicon struct Detec- Size Assay Region Fwd-P Rev-P tion [bp] 1 Region HemB- HemB- HemB- 81 1 FWD-A REV-A Probe-A (SEQ ID (SEQ ID (SEQ ID NO: 33) NO: 39) NO: 47) 2 Region HemB- HemB- HemB- 107 2 FWD-B REV-C Probe-B (SEQ ID (SEQ ID (SEQ ID NO: 35) NO: 43) NO: 51) 3 HemB- HemB- HemB- 82 FWD-C REV-B Probe-D (SEQ ID (SEQID (SEQ ID NO: 37) NO: 41) NO: 59) 4 HemB- HemB- HemB- 101 FWD-B REV-D Probe-C (SEQ ID (SEQ ID (SEQ ID NO: 35) NO: 45) NO: 55) 5 Region HemB- HemB- HemB- 140 3 FWD-D REV-F Probe-E (SEQ ID (SEQ ID (SEQ ID NO: 81) NO: 87) NO: 89) Ampl. effi- 62500 15625 3906.25 976.56 cien- Assay copies copies copies copies slope cy 1 22.6 24.6 26.7 28.6 −3.32 100.1 2 23.1 25.2 27.2 29.1 −3.31 100.5 3 24.1 26.1 28.2 30.1 −3.35 98.9 4 23.2 25.1 27.3 29.2 −3.33 99.8 5 23.0 25.1 26.8 29.2 −3.36 98.6
TABLE-US-00022 TABLE 17 Primary Screening of Certain Multiplexed Assay Results of Exemplary HEM-B AAV Constructs Assay Set DNA Source Fluorescence Channel Assay Primers Target 1 Exemplary HemB AAV HemB AAV probe specific Exemplary HemB AAV Construct Construct 2 IC-dsDNA IC probe specific IC target 3 3 Exemplary HemB AAV HemB AAV probe specific Exemplary HemB AAV 4 Construct and IC probe specific Construct and IC target IC-dsDNA 3 5 Exemplary HemB AAV HemB AAV probe specific Exemplary HemB AAV 6 Construct IC probe specific Construct and IC target 7 Exemplary HemB AAV HemB AAV probe specific 3 8 Construct IC probe specific 9 Exemplary HemB AAV HemB AAV probe specific 10 Construct and IC probe specific IC-dsDNA 62500 15625 3906.25 976.56 244.1 Ampl. Assay Set copies copies copies copies copies slope efficiency 1 22.4 24.5 26.9 29.0 30.6 −3.5 94.1 2 19.2 21.2 23.3 25.3 27.5 −3.4 96.5 3 22.1 24.2 26.4 28.4 30.5 −3.5 93.3 4 19.0 21.1 23.2 25.2 27.2 −3.4 97.0 5 22.0 24.1 26.4 28.5 30.3 −3.5 93.9 6 N/A N/A N/A N/A N/A N/A N/A 7 N/A N/A N/A N/A N/A N/A N/A 8 19.6 21.5 23.5 25.5 27.4 −3.3 102.2 9 22.1 24.3 25.8 28.5 30.5 −3.5 94.6 10 19.5 21.5 23.5 25.4 27.7 −3.4 98.7
TABLE-US-00023 TABLE 18 Certain Primer/Probe Sets for Amplifying and Quantifying Exemplary Hem-B AAV Constructs Exemplary Hem-B AAV Construct Assay Region Primer/Probe Set Slope Fluorescence CT N Region 1 Set 10 −3.2 4.0 24.8 10 (SEQ ID NOs: 33, 39, and 47) Region 2 Set 6 −3.37 4.4 22.8 10 (SEQ ID NOs: 35, 45, and 55) Region 3 Set 9 −3.31 4.5 23.2 10 (SEQ ID NOs: 81, 87, and 89)
[0258] Statistical Analysis
[0259] Accuracy—Assay accuracy was determined by calculating the average log recovery, defined as the average difference between the observed mean log quantitation and the log input concentration for levels tested (expected).
[0260] Linearity—The assay linearity was assessed by fitting a linear regression model as follows
Log 10(Quantitation)=β0+β1×Log 10(Input Concentration)+ε
[0261] Where β0 is intercept, β1 is slope and c is the random error term. The coefficient of determination R.sup.2 was calculated based on this model. Linearity is established if R.sup.2 is above 0.95. Accuracy and linearity analysis was performed using R (Version 3.5.1 2018-07-02, https://cran.rproject.org/)
[0262] Precision—Assay within-laboratory reproducibility, or precision, was estimated using the following variance components model for each concentration level,
Quantitation=intercept+instrument+run+within-run (error)
[0263] Where instrument, run, and within-run errors were treated as random effects. Variance components were estimated via a random effect model. The total variance, δ.sup.2, which accounted for all between-instrument, between-run, and within-run effects, was estimated as the sum of the individual variance components. The measure of reproducibility is the percent coefficient of variation (% CV), or the total variance divided by the mean. Variance components were analyzed in JMP (Version 13.1, SAS Institute) using REML (Restricted Maximum Likelihood) model (https://www.jmp.com/support/help/14-2/restricted-maximumlikelihood-reml-model.shtml).
[0264] Specificity—Assay specificity was calculated as one minus the ratio of false positive samples to all unspiked (negative) samples.
[0265] Analytical Sensitivity—The Lower Limit of Quantitation (LLoQ) is the lowest input concentration that meets all above criteria and at which detection is at least 95%.
[0266] RESULTS:
[0267] qPCR Performance Summary—The lower limit of quantitation (LLoQ) of qPCR is 31 copies of exemplary Hem-B AAV construct viral DNA in the background of 1 μg genomic DNA.
[0268] Shedding Assay Performance Summary—The shedding assay provides a quantitative result for plasma, PBMC, saliva, semen, stool and urine samples within corresponding assay ranges. The shedding assay detection range is summarized in Table 19. If the titer was detected to be above the upper quantitation limit, the sample was reported as AQL (above quantitation limit); if the titer was determined to be below the lower quantitation limit, the sample was be reported as BQL (below quantitation limit); and if the titer was within the quantitation range, a numeric concentration was reported.
TABLE-US-00024 TABLE 19 Shedding assay detection range summary Sample Lower Limit of Quantitation Upper Limit of Quantitation Type (LLoQ) (ULoQ) Plasma 1440 vg/mL 1.0E+08 vg/mL PBMC 316 vg/1 μg genomic DNA 4.7E+07 vg/1 μg genomic DNA Saliva 6172 vg/mL 1.3E+09 vg/mL Semen 6037 vg/mL 1.5E+09 vg/mL Stool 25 vg/mg 1.4E+07 vg/mg stool Urine 5494 vg/mL 1.4E+09 vg/mL
[0269] DNase Treatment Performance Summary—MNase was the chosen nuclease for the DNase treatment. MNase acts as a potent DNA and RNA endonuclease, cleaving double-stranded DNA(dsDNA), single-stranded DNA(ssDNA) and RNA. MNase is known to be more potent than DNase I (MNase at 2,000,000 units/mL and DNase I at 2,000 units/mL are commercially available through New England Biolabs). Incomplete digestion of unprotected DNA was observed with DNase I (up to 250 units, limited by the stock concentration) in reaction buffer system. In comparison, treatment with 4,000 units of MNase eliminated the unprotected deoxynucleic acid (both dsDNA and ssDNA) and did not decrease exemplary Hem-B AAV construct recovery from intact viral particles, in all sample types tested.
[0270] qPCR Performance—The FDA guidance requires qPCR to be able to detect<50 copies of vector/1 μg genomic DNA with 95% confidence. The qPCR quantitation limit was determined by using a panel of linearized plasmid containing the exemplary Hem-B AAV construct viral genome spiked into human genomic DNA at different concentrations. Linearized plasmid at different concentrations was tested with different numbers of replicates to assess the quantification limits. N=75 was chosen for the lowest concentration to ensure a 95% detection rate with enough confidence. The lower limit of quantification (LLoQ) for qPCR was determined to be 31 copies of viral genome/1 μg genomic DNA based on the criteria of ≥95% detection, ≤20% variability and within 0.125 LOG of difference from the target value (summarized in Table 20, exceeding the FDA guidance requirement.
TABLE-US-00025 TABLE 20 Performance Evaluation Report Summary Accuracy Precision Observed Expected No. of No. Detection (LogObs-LogExp, (<20% (vg/1 μg gDNA) (vg/1 μg gDNA) Reps Detected Rate Within ± 0.125LOG) CV) 455 500 22 22 100.0% −0.041 10.10% 42 50 12 12 100.0% −0.075 14.13% 31 37.5 19 19 100.0% −0.081 14.74% 19.4 25.0 75 75 100.0% −0.110 20.47%
[0271] The standalone sample preparation 1.0 (Siemens Healthineers SASP 1.0) system is a scalable, automated, and optimized multi-sample type preparation system that allows the barcode tracking for samples and controls. SASP 1.0 kits had increased precision with semen samples in comparison to SASP 1.2 kit, and the optimized system allows batch processing of all six biological sample types within the same run, achieving high synergy for the shedding assay.
[0272] Specificity—The specificity of the exemplary Hem-B AAV construct shedding assay was determined with 32 biological replicates each, using normal human plasma, PBMC, urine, saliva, semen and stool samples without any viral spike. In all sample types, 32/32 replicates were below quantitation limit, and the specificity was 100% in all sample types tested.
[0273] Analytical Sensitivity—During the value assignment process, linearized plasmid was used as standards in parallel with viral material diluted in a virus storage buffer processed through the extraction and qPCR. The viral stock titer was determined to be 1.53E+13 vg/mL based on the linearized plasmid standards. Performance evaluation was done in two steps. In Step 1, for each sample type, a 12-replicate panel was tested to estimate the Lower Limit of Quantitation (LLoQ); in Step 2, up to 36 biological replicates near the estimated LLoQ level were utilized to further refine LLoQ. The lowest sample concentration that demonstrated to have≤30% CV and within ±0.5 LOG of bias was determined as the LLoQ for that sample type. Exemplary Hem-B AAV construct shedding assay analytical sensitivity for each sample type is summarized in Tables 21-26 below.
TABLE-US-00026 TABLE 21 Analytical Sensitivity in Plasma Concen- No. LLoQ tration of No. Detection (LogObs- Total [95% CI] (vg/mL) Reps Detected Rate LogExp) % CV (vg/mL) 1.0E+09 12 12 100.0% 0.013 14.4% 1440 1.1E+07 12 12 100.0% 0.063 12.9% [662-2218] 1.2E+05 12 12 100.0% 0.086 9.9% 1.4E+03 36 36 100.0% 0.167 27.6% (LLoQ) 1.1E+03 36 36 100.0% 0.146 35.1% 1.1E+03 12 12 100.0% 0.032 26.9% 6.9E+02 36 36 100.0% 0.102 44.4% 5.3E+02 36 36 100.0% 0.087 39.0% 5.1E+02 12 12 100.0% −0.080 31.7% 3.4E+02 12 11 91.7% 0.080 21.7% 8.4E+01 12 5 41.7% 0.407 42.9% 6.8E+01 12 2 16.7% 0.614 61.0%
TABLE-US-00027 TABLE 22 Analytical Sensitivity in PBMC* Concen- LLoQ tration No. [95% CI] (vg/1 μg of No. Detection (LogObs- Total (vg/1 μg gDNA) Reps Detected Rate LogExp) % CV gDNA) 4.7E+07 12 12 100.0% 0.169 10.2% 316 5.0E+05 12 12 100.0% 0.197 11.4% [176-455] 5.5E+03 12 12 100.0% 0.256 16.8% 5.3E+02 36 36 100.0% 0.241 21.9% 4.5E+02 12 12 100.0% 0.168 15.3% 3.2E+02 36 36 100.0% 0.231 22.5% (LLoQ) 1.4E+02 36 36 100.0% 0.188 32.5% 9.4E+01 36 36 100.0% 0.175 32.4% 7.3E+01 36 36 100.0% 0.187 39.4% 6.2E+01 12 12 100.0% 0.129 36.1% 3.4E+01 12 12 100.0% 0.139 45.1% 1.3E+01 12 9 75.0% 0.255 85.1% 9.8E+00 12 10 83.3% 0.438 62.8% *PBMCs were re-suspended in cell freezing media at the concentration of 5E+6 cells/mL.
TABLE-US-00028 TABLE 23 Analytical Sensitivity in Saliva Concen- No. LLoQ tration of No. Detection (LogObs- Total [95% CI] (vg/mL) Reps Detected Rate LogExp) % CV (vg/mL) 1.3E+09 12 12 100.0% 0.092 6.4% 6172 1.4E+07 12 12 100.0% 0.144 4.3% [3045-9299] 1.5E+05 12 12 100.0% 0.171 9.2% 2.0E+04 36 36 100.0% 0.128 17.0% 2.0E+04 12 12 100.0% 0.124 15.9% 1.3E+04 36 36 100.0% 0.116 22.5% 1.1E+04 12 12 100.0% 0.145 18.8% 9.6E+03 36 36 100.0% 0.133 19.6% 6.2E+03 36 36 100.0% 0.087 25.9% (LLoQ) 2.0E+03 36 36 100.0% 0.060 37.5% 1.2E+03 12 12 100.0% 0.076 37.0% 4.2E+02 12 3 25.0% 0.466 69.1% 2.6E+02 12 5 41.7% 0.572 13.1%
TABLE-US-00029 TABLE 24 Analytical Sensitivity in Semen Concen- No. LLoQ tration of No. Detection (LogObs- Total [95% CI] (vg/mL) Reps Detected Rate LogExp) % CV (vg/mL) 1.5E+09 12 12 100.0% 0.223 8.9% 6037 1.4E+07 12 12 100.0% 0.225 14.2% [3599- 1.4E+05 12 12 100.0% 0.255 22.8% 8475] 2.3E+04 12 12 100.0% 0.259 28.2% 2.3E+04 36 36 100.0% 0.388 27.3% 2.0E+04 36 36 100.0% 0.448 24.9% 1.8E+04 36 36 100.0% 0.450 26.9% 1.6E+04 33 33 100.0% 0.491 24.8% 6.0E+03 12 12 100.0% 0.295 20.6% (LLoQ) 4.2E+03 12 12 100.0% 0.442 34.8% 1.8E+03 12 12 100.0% 0.522 95.7% 1.3E+03 12 6 50.0% 0.705 50.4%
TABLE-US-00030 TABLE 25 Analytical Sensitivity in Stool** Concen- LLoQ tration No. [95% CI] (vg/mg of No. Detection (LogObs- Total (vg/mg stool) Reps Detected Rate LogExp) % CV stool) 1.4E+07 12 12 100.0% 0.115 14.2% 25 1.5E+05 12 12 100.0% 0.140 11.6% [13-37] 1.4E+03 12 12 100.0% 0.144 13.1% 1.4E+02 11† 11 100.0% 0.138 12.6% 3.9E+01 36 36 100.0% 0.109 28.9% 2.5E+01 12 12 100.0% 0.104 24.8% (LLoQ) 2.4E+01 36 36 100.0% 0.070 38.1% 2.0E+01 36 36 100.0% 0.109 40.8% 1.4E+01 12 12 100.0% 0.131 31.9% 1.3E+01 36 36 100.0% 0.104 42.8% 6.6E+00 12 11 91.7% 0.168 49.0% 3.3E+00 12 9 75.0% 0.173 53.5% **Stool was suspended in 10 volume (w:v) of 1X PBS before extraction. †1 sample lost due to clogging
TABLE-US-00031 TABLE 26 Analytical Sensitivity in Urine Concen- No. LLoQ tration of No. Detection (LogObs- Total [95% CI] (vg/mL) Reps Detected Rate LogExp) % CV (vg/mL) 1.4E+09 12 12 100.0% 0.134 19.0% 5494 1.4E+07 12 12 100.0% 0.135 9.5% [2465-8524] 1.5E+05 12 12 100.0% 0.187 18.7% 1.1E+04 36 36 100.0% 0.151 17.4% 1.1E+04 12 12 100.0% 0.142 27.0% 5.5E+03 36 36 100.0% 0.132 28.1% (LLoQ) 4.1E+03 36 36 100.0% 0.130 30.1% 2.8E+03 36 36 100.0% 0.147 30.7% 2.3E+03 12 12 100.0% 0.062 40.3% 1.3E+03 12 11 91.7% 0.100 31.2% 6.2E+02 12 9 75.0% 0.245 39.6% 5.9E+02 12 5 41.7% 0.520 51.7%
[0274] Accuracy—Titer data were considered to be log-normally distributed and were analyzed following log 10 transformation. Exemplary Hem-B AAV construct shedding assay accuracy was determined using the same sample panels described above for analytical sensitivity. Accuracy was calculated by the difference between observed value and the expected value after log transformation, and the target requirement was with ±0.5 log bias of expected value across the quantitative range. Exemplary Hem-B AAV construct shedding assay linearity and accuracy for each sample type was summarized and presented in Tables 27-32 and
TABLE-US-00032 TABLE 27 Accuracy in Plasma Observed Expected Concentration LogQTY LogQTY No. of Log Difference (vg/mL) (Log vg/mL) (Log vg/mL) Reps (LogObs-LogExp) 1.0E+09 9.00 8.99 12 0.01 1.1E+07 7.06 7.00 12 0.06 1.2E+05 5.08 5.00 12 0.09 1.4E+03(LLoQ) 3.16 2.99 36 0.17 1.1E+03 3.03 2.88 36 0.15 1.1E+03 3.02 2.99 12 0.03 6.9E+02 2.84 2.73 36 0.10 5.3E+02 2.72 2.64 36 0.09 5.1E+02 2.71 2.79 12 −0.08 3.4E+02 2.54 2.46 12 0.08 8.4E+01 1.92 1.52 12 0.41 6.8E+01 1.83 1.22 12 0.61
TABLE-US-00033 TABLE 28 Accuracy in PBMC Observed Expected LogQTY LogQTY Concentration (Log vg/μg (Log vg/μg No. of Log Difference (vg/1 μg gDNA) gDNA) gDNA) Reps (LogObs-LogExp) 4.7E+07 7.67 7.50 12 0.17 5.0E+05 5.69 5.50 12 0.20 5.5E+03 3.74 3.48 12 0.26 5.3E+02 2.73 2.49 36 0.24 4.5E+02 2.65 2.48 12 0.17 3.2E+02 (LLoQ) 2.50 2.27 36 0.23 1.4E+02 2.15 1.97 36 0.19 9.4E+01 1.97 1.79 36 0.17 7.3E+01 1.86 1.67 36 0.19 6.2E+01 1.79 1.66 12 0.13 3.4E+01 1.53 1.39 12 0.14 1.3E+01 1.11 0.86 12 0.25 9.8E+00 0.99 0.55 12 0.44 *PBMCs were re-suspended in cell freezing media at the concentration of 5E+6 cells/mL.
TABLE-US-00034 TABLE 29 Accuracy in Saliva Observed Expected Concentration LogQTY LogQTY No. of Log Difference (vg/mL) (Log vg/mL) (Log vg/mL) Reps (LogObs-LogExp) 1.3E+09 9.11 9.01 12 0.09 1.4E+07 7.15 7.01 12 0.14 1.5E+05 5.16 4.99 12 0.17 2.0E+04 4.31 4.18 36 0.13 2.0E+04 4.29 4.17 12 0.12 1.3E+04 4.12 4.00 36 0.12 1.1E+04 4.04 3.90 12 0.15 9.6E+03 3.98 3.85 36 0.13 6.2E+03 (LLoQ) 3.79 3.70 36 0.09 2.0E+03 3.30 3.24 36 0.06 1.2E+03 3.08 3.00 12 0.08 4.2E+02 2.62 2.16 12 0.47 2.6E+02 2.42 1.84 12 0.57
TABLE-US-00035 TABLE 30 Accuracy in Semen Observed Expected Concentration LogQTY LogQTY No. of Log Difference (vg/mL) (Log vg/mL) (Log vg/mL) Reps (LogObs-LogExp) 1.5E+09 9.17 8.94 12 0.22 1.4E+07 7.15 6.93 12 0.22 1.4E+05 5.16 4.91 12 0.25 2.3E+04 4.36 4.10 12 0.26 2.3E+04 4.35 3.97 36 0.39 2.0E+04 4.31 3.86 36 0.45 1.8E+04 4.24 3.79 36 0.45 1.6E+04 4.20 3.71 33 0.49 6.0E+03 (LLoQ) 3.78 3.49 12 0.29 4.2E+03 3.62 3.18 12 0.44 1.8E+03 3.25 2.73 12 0.52 1.3E+03 3.13 2.42 12 0.71
TABLE-US-00036 TABLE 31 Accuracy in Stool Concentration Observed LogQTY Expected LogQTY Log Difference (vg/mg) (Log vg/mg) (Log vg/mg) No. of Reps (LogObs-LogExp) 1.4E+07 7.14 7.02 12 0.11 1.5E+05 5.16 5.02 12 0.14 1.4E+03 3.15 3.01 12 0.14 1.4E+02 2.14 2.01 11† 0.14 3.9E+01 1.59 1.48 36 0.11 2.5E+01 (LLoQ) 1.4 1.3 12 0.10 2.4E+01 1.38 1.31 36 0.07 2.0E+01 1.29 1.18 36 0.11 1.4E+01 1.14 1.01 12 0.13 1.3E+01 1.11 1.01 36 0.10 6.6E+00 0.82 0.65 12 0.17 3.3E+00 0.52 0.35 12 0.17 **Stool was suspended in 10 volume (w:v) of 1X PBS before extraction, †1 sample lost due to clogging
TABLE-US-00037 TABLE 32 Accuracy in Urine Concentration Observed LogQTY Expected LogQTY Log Difference (vg/mL) (Log vg/mL) (Log vg/mL) No. of Reps (LogObs-LogExp) 1.4E+09 9.15 9.02 12 0.13 1.4E+07 7.14 7.01 12 0.13 1.5E+05 5.18 4.99 12 0.19 1.1E+04 4.06 3.91 36 0.15 1.1E+04 4.04 3.90 12 0.14 5.5E+03 (LLoQ) 3.74 3.61 36 0.13 4.1E+03 3.61 3.48 36 0.13 2.8E+03 3.45 3.31 36 0.15 2.3E+03 3.36 3.30 12 0.06 1.3E+03 3.10 3.00 12 0.10 6.2E+02 2.79 2.55 12 0.24 5.9E+02 2.77 2.25 12 0.52
[0275] Precision—Assay within-laboratory precision was determined using the same panels described in analytical sensitivity (described above). Each panel was tested with the same reagent lot using two instruments on multiple days. Target requirement was ≤30% CV throughout the quantitative range. Exemplary Hem-B AAV construct shedding assay within-laboratory precision for each sample type was summarized and is presented in Tables 33-38.
TABLE-US-00038 TABLE 33 Components of Variation for Plasm Panel Within Between Between Concentration No. of Run Run Instrument Overall (vg/mL) Reps (% CV) (% CV) (% CV) (% CV) 1.0E+09 12 6.4% 12.7% 12.3% 14.4% 1.1E+07 12 8.1% 5.4% 11.9% 12.9% 1.2E+05 12 8.0% 3.1% 5.9% 9.9% 1.4E+03 (LLoQ) 36 21.3% 15.0% 14.1% 27.6% 1.1E+03 36 26.4% 15.8% 20.8% 35.1% 1.1E+03 12 24.7% 13.7% 16.2% 26.9% 6.9E+02 36 43.9% 22.1% 15.1% 44.4% 5.3E+02 36 38.9% 2.9% 14.1% 39.0% 5.1E+02 12 25.1% 2.4% 21.3% 31.7% 3.4E+02 12 Below assay detection range 8.4E+01 12 6.8E+01 12
TABLE-US-00039 TABLE 34 Components of Variation for PBMC* Panel Within Between Between Concentration No. of Run Run Instrument Overall (vg/μg gDNA) Reps (% CV) (% CV) (% CV) (% CV) 4.7E+07 12 5.5% 5.9% 10.7% 10.2% 5.0E+05 12 7.7% 7.4% 11.1% 11.4% 5.5E+03 12 16.6% 6.4% 5.9% 16.8% 5.3E+02 36 16.1% 7.9% 14.1% 21.9% 4.5E+02 12 14.9% 3.2% 7.8% 15.3% 3.2E+02 (LLoQ) 36 17.2% 6.9% 15.8% 22.5% 1.4E+02 36 28.8% 16.0% 10.2% 32.5% 9.4E+01 36 32.4% 7.6% 0.7% 32.4% 7.3E+01 36 31.5% 19.5% 19.7% 39.4% 6.2E+01 12 32.0% 22.6% 26.2% 36.1% 3.4E+01 12 42.6% 25.5% 26.4% 45.1% 1.3E+01 12 Below assay detection range 9.8E+00 12 *PBMCs were re-suspended in cell freezing media at the concentration of 5E+6 cells/mL.
TABLE-US-00040 TABLE 35 Components of Variation for Saliva Panel Within Between Between Concentration No. of Run Run Instrument Overall (vg/mL) Reps (% CV) (% CV) (% CV) (% CV) 1.3E+09 12 3.5% 6.3% 2.9% 6.4% 1.4E+07 12 4.2% 1.1% 0.2% 4.3% 1.5E+05 12 7.8% 7.9% 3.5% 9.2% 2.0E+04 36 11.7% 4.2% 13.0% 17.0% 2.0E+04 12 13.5% 9.0% 5.4% 15.9% 1.3E+04 36 15.5% 12.7% 14.0% 22.5% 1.1E+04 12 14.4% 9.4% 15.4% 18.8% 9.6E+03 36 18.0% 8.2% 4.9% 19.6% 6.2E+03 (LLoQ) 36 24.9% 13.2% 6.4% 25.9% 2.0E+03 36 34.3% 16.1% 10.0% 37.5% 1.2E+03 12 31.9% 23.7% 28.4% 37.0% 4.2E+02 12 Below assay detection range 2.6E+02 12
TABLE-US-00041 TABLE 36 Components of Variation for Semen Panel Within Between Between Concentration No. of Run Run Instrument Overall (vg/mL) Reps (% CV) (% CV) (% CV) (% CV) 1.5E+09 12 6.8% 6.4% 8.3% 8.9% 1.4E+07 12 11.1% 7.7% 11.7% 14.2% 1.4E+05 12 9.4% 2.5% 22.8% 22.8% 2.3E+04 12 22.5% 10.0% 20.4% 28.2% 2.3E+04 36 22.6% 8.8% 17.1% 27.3% 2.0E+04 36 22.4% 12.1% 6.7% 24.9% 1.8E+04 36 23.8% 16.0% 4.9% 26.9% 1.6E+04 33 24.0% 8.1% 2.3% 24.8% 6.0E+03 (LLoQ) 12 20.4% 7.7% 9.3% 20.6% 4.2E+03 12 32.4% 16.5% 19.4% 34.8% 1.8E+03 12 85.8% 57.1% 66.0% 95.7% 1.3E+03 12 Below assay detection range
TABLE-US-00042 TABLE 37 Components of Variation for Stool** Panel Within Between Between Concentration No. of Run Run Instrument Overall (vg/mg stool) Reps (% CV) (% CV) (% CV) (% CV) 1.4E+07 12 4.8% 7.4% 13.4% 14.2% 1.5E+05 12 8.3% 7.9% 6.1% 11.6% 1.4E+03 12 8.1% 5.7% 10.4% 13.1% 1.4E+02 11† 10.8% 4.5% 6.3% 12.6% 3.9E+01 36 27.5% 11.8% 2.7% 28.9% 2.5E+01 (LLoQ) 12 24.2% 4.8% 13.5% 24.8% 2.4E+01 36 34.2% 4.8% 17.6% 38.1% 2.0E+01 36 30.8% 10.9% 26.4% 40.8% 1.4E+01 12 31.7% 13.8% 14.2% 31.9% 1.3E+01 36 40.1% 11.8% 17.6% 42.8% 6.6E+00 12 Below assay detection range 3.3E+00 12 **Stool was suspended in 10 volume (w:v) of 1X PBS before extraction. †1 sample lost due to clogging
TABLE-US-00043 TABLE 38 Components of Variation for Urine Panel Within Between Between Concentration No. of Run Run Instrument Overall (vg/mL) Reps (% CV) (% CV) (% CV) (% CV) 1.4E+09 12 15.6% 11.9% 7.1% 19.0% 1.4E+07 12 7.7% 5.9% 3.8% 9.5% 1.5E+05 12 10.6% 7.7% 18.2% 18.7% 1.1E+04 36 14.9% 10.7% 4.7% 17.4% 1.1E+04 12 24.5% 16.7% 5.0% 27.0% 5.5E+03 (LLoQ) 36 25.3% 5.4% 13.3% 28.1% 4.1E+03 36 28.5% 21.6% 12.5% 30.1% 2.8E+03 36 30.2% 7.9% 8.3% 30.7% 2.3E+03 12 37.8% 16.1% 20.4% 40.3% 1.3E+03 12 23.4% 21.1% 26.5% 31.2% 6.2E+02 12 Below assay detection range 5.9E+02 12
[0276] Individual Variability—Individual variability was assessed with five different donors for each sample type spiked with 1.0E+07 vg/mL exemplary Hem-B AAV construct (post 5E+6 cells/mL suspension of PBMCs and 1:10(w:v) suspension of stool). Three biological replicates for each condition were performed for the assessment. Individual variability was observed to be in the same range of assay reproducibility/precision (% CV≤30% and within ±0.5 LOG difference), data presented in Table 39, and
TABLE-US-00044 TABLE 39 Individual Variability Sample Type Donors* Mean Titer Log Mean Titer % CV Plasma 1 1.17E+07 7.07 12.5% (vg/mL) 2 1.08E+07 7.03 2.6% 3 1.01E+07 7.00 13.9% 4 1.02E+07 7.01 12.8% 5 1.03E+07 7.01 9.9% PBMC 1 4.71E+05 5.67 11.4% (vg/μg gDNA) 2 4.62E+05 5.66 8.1% 3 5.02E+05 5.70 7.7% 4 4.74E+05 5.68 13.5% 5 5.31E+05 5.73 3.5% Saliva 1 1.45E+07 7.16 4.5% (vg/mL) 2 1.31E+07 7.12 8.5% 3 1.32E+07 7.12 9.5% 4 1.29E+07 7.11 6.7% 5 1.23E+07 7.09 2.5% Semen 1 1.86E+07 7.27 5.9% (vg/mL) 2 1.69E+07 7.23 1.0% 3 1.63E+07 7.21 7.2% 4 1.42E+07 7.15 2.1% 5 1.56E+07 7.19 0.5% Stool 1 1.54E+05 5.19 5.2% (vg/mg) 2 1.28E+05 5.11 5.2% 3 1.49E+05 5.17 8.7% 4 1.23E+05 5.09 8.2% 5 1.20E+05 5.08 3.0% Urine 1 1.19E+07 7.07 0.7% (vg/mL) 2 1.14E+07 7.06 8.5% 3 1.49E+07 7.17 8.5% 4 1.29E+07 7.11 4.9% 5 1.16E+07 7.07 16.6% *Donors for different sample types did not match; each sample types had different donors
[0277] DNase Treatment—MNase was the chosen nuclease for DNase treatment. To assess the efficiency of nuclease activity, saliva, semen, urine and 1X PBS suspended stool was spiked with three types of deoxynucleic acid templates: unprotected double-stranded DNA (dsDNA-AAVR), single-stranded DNA (ssDNA-GFP), and encapsulated viral DNA (rAAV—exemplary Hem-B AAV construct). dsDNA-AAVR and ssDNA-GFP were spiked in parallel with rAAV-exemplary Hem-B AAV construct, all at the concentration of 1.0E+07 copies/mL, to assess the efficiency of nuclease activity. MNase treatment was done in 3 replicates with 4,000 unit of MNase per sample at 37° C. for 30 min, and stopped by the addition of EDTA. Siemens storage buffer (SSB2) was spiked in the same manner, without MNase treatment, to serve as baseline for recovery calculation. After the MNase reaction was complete, DNA extraction was performed in accordance with methods known in the art.
[0278] Strong endogenous nuclease activity toward unprotected deoxynucleic acids, both dsDNA and ssDNA, was observed in PBS suspended stool samples. Moderate endogenous nuclease activity toward ssDNA was also observed in saliva, and to a lesser extent in semen and urine samples. MNase treatment in all sample types depleted unprotected dsDNA and ssDNA templates (>97.5% reduction) without decreasing rAAV recovery. Exemplary Hem-B AAV construct MNase treatment results are summarized in
[0279] The effect of MNase treatment on intact viral DNA recovery was further assessed in all sample types at a concentration near the LLoQ, with a minimum of 12 replicates. No significant difference was observed between MNase treated or untreated in all sample types, results are summarized in
TABLE-US-00045 TABLE 40 Summary of rAAV, dsDNA and ssDNA Percent Recovery with or without MNase Treatment in Tested Sample Types Percent Recovery (Relative to Spiked Untreated Siemens Storage Buffer) rAAV (HemB AAV, dsDNA (AAVR, Sample protected) unprotected) ssDNA (GFP, unprotected) type Not Treated +MNase Not Treated +MNase Not Treated +MNase Saliva 117 ± 4% 125 ± 5% 37 ± 2% 2 ± 1% 11 ± 1% < 1% Semen 95 ± 18% 88 ± 2% 77 ± 8% <1% 32 ± 1% < 1% Stool 128 ± 2% 164 ± 2% ND ND <1% < 1% Urine 141 ± 4% 138 ± 2% 165 ± 19% ND 33 ± 1% ND ND = not detected by qPCR
TABLE-US-00046 TABLE 41 Summary of Exemplary HemB AAV Construct Recovery with or without MNase Treatment in Each Sample Type near LLoQ HemB AAV Sample type Not Treated +MNase P value Saliva (LOG vg/mL) 3.78 ± 0.11 3.82 ± 0.10 0.33 Semen (LOG vg/mL) 4.00 ± 0.08 3.95 ± 0.23 0.75 Urine (LOG vg/mL) 3.69 ± 0.08 3.67 ± 0.11 0.69 Stool (LOG vg/mg) 1.37 ± 0.16 1.48 ± 0.11 0.09
[0280] Analyte Stability—Stability of exemplary Hem-B AAV construct was analyzed in both unprocessed whole blood and processed specimens—plasma, PBMCs, urine, saliva, semen and stool, under different conditions.
[0281] Room Temperature Stability of Unprocessed Specimen—Whole Blood—Stability of exemplary Hem-B AAV construct in whole blood stored at RT was determined using blood freshly drawn from 3 individual donors and spiked with 1.0E+07 vg/mL of exemplary Hem-B AAV construct. Plasma was isolated after storing whole blood at RT for indicated time and kept in −80° C. until the last time point was collected for plasma shedding assay analysis. Three biological replicates were tested for each condition. Individual to individual variability was observed; one of the donors showed a 0.3-0.7 LOG decrease after the 24 hr RT incubation. Stability results are summarized in
TABLE-US-00047 TABLE 42 Stability of Exemplary HemB AAV Construct in Whole Blood Stored at RT Log Mean Donors Time(h) Mean Titer Titer % CV Donor1 0 6.85E+06 6.83 23.6% (vg/mL) 4 6.38E+06 6.80 2.8% 8 6.85E+06 6.84 4.5% 24 7.74E+06 6.89 7.4% 48 8.98E+06 6.95 4.7% 0 6.85E+06 6.83 1.3% Donor2 4 6.38E+06 6.73 1.3% (vg/mL) 8 6.85E+06 6.55 1.3% 24 7.74E+06 6.13 0.9% 48 8.98E+06 6.51 0.8% Donor3 0 5.54E+06 6.74 30.1% (vg/mL) 4 6.18E+06 6.79 6.5% 8 6.73E+06 6.83 1.5% 24 7.30E+06 6.86 3.3% 48 8.61E+06 6.93 4.0%
[0282] Room Temperature Stability of Unprocessed Specimen—Plasma, PBMCs, Saliva, Semen, Urine and Stool—Stability of exemplary Hem-B AAV construct virus plasma, PBMCs, saliva, semen, stool and urine, stored at RT was assessed at different time points. Samples pooled from five donors were spiked with 1.0E+05 vg/mL exemplary Hem-B AAV construct (post 5E+6 cells/mL suspension of PBMCs and 1:10 (w:v) suspension of stool), and three biological replicates per condition were tested at indicated time points. Exemplary Hem-B AAV construct viral genome titer was within ±0.5 LOG of the titer determined at time 0 for up to 48 hours except stool, which was stable up to 24 hours. See results summary in
TABLE-US-00048 TABLE 43 Stability Samples Stored at RT Sample Type Time (h) Mean Titer Log Mean Titer % CV Plasma 0 7.19E+04 4.86 15.0% (vg/mL) 4 7.42E+04 4.87 14.8% 8 7.80E+04 4.89 16.9% 24 7.20E+04 4.86 14.3% 0 5.90E+04 4.77 7.2% 48 6.00E+04 4.78 7.9% PBMC 0 2.55E+02 2.41 7.7% (vg/μg gDNA) 4 3.80E+02 2.58 9.4% 8 4.23E+02 2.63 4.7% 24 4.60E+02 2.66 3.8% 0 3.28E+02 2.52 22.9% 48 4.81E+02 2.68 20.0% Saliva 0 2.81E+04 4.45 9.6% (vg/mL) 4 3.02E+04 4.48 17.0% 8 2.77E+04 4.44 7.2% 24 2.59E+04 4.41 11.4% 0 2.61E+04 4.42 5.6% 48 2.41E+04 4.38 6.8% Semen 0 1.59E+04 4.20 17.3% (vg/mL) 4 2.03E+04 4.31 10.9% 8 1.98E+04 4.30 7.3% 24 2.01E+04 4.30 4.6% 0 1.44E+04 4.16 2.6% 48 1.68E+04 4.23 12.5% Stool 0 3.53E+02 2.55 6.4% (vg/mg) 4 3.18E+02 2.50 7.7% 8 3.45E+02 2.54 4.4% 24 3.20E+02 2.51 5.0% 0 3.19E+02 2.50 5.5% 48 1.12E+02 2.05 15.0% Urine 0 3.11E+04 4.49 9.9% (vg/mL) 4 2.21E+04 4.34 8.7% 8 2.79E+04 4.45 14.5% 24 2.83E+04 4.45 14.2% 0 2.98E+04 4.47 2.7% 48 2.86E+04 4.46 2.9%
[0283] Freeze/Thaw Stability of Plasma, PBMCs, Saliva, Semen, Urine and Stool—Stability of exemplary Hem-B AAV construct virus in plasma, PBMCs, urine, saliva, semen and stool for up to 4 freeze/thaw cycles was assessed. Samples pooled from five donors were spiked at 1.0E+05 vg/mL (post 5E+6 cells/mL suspension of PBMCs and 1:10 (w:v) suspension of stool), and three biological replicates per condition were tested. Exemplary Hem-B AAV construct viral genome titer was within ±0.5 LOG of starting condition up to 4 freeze-thaw cycles. See results summary in
TABLE-US-00049 TABLE 44 Summary of Sample Freeze/Thaw Stability Freeze Thaw Sample Type Cycles Mean Titer Log Mean Titer % CV Plasma 1 6.02E+04 4.78 8.0% (vg/mL) 2 5.87E+04 4.77 2.4% 3 6.16E+04 4.79 5.2% 4 6.65E+04 4.82 3.2% PBMC 1 7.76E+02 2.89 15.5% (vg/μg gDNA) 2 6.58E+02 2.82 12.3% 3 9.29E+02 2.97 2.3% 4 8.15E+02 2.91 11.2% Saliva 1 2.68E+04 4.43 4.1% (vg/mL) 2 2.42E+04 4.38 7.0% 3 2.52E+04 4.40 9.6% 4 2.27E+04 4.36 11.6% Semen 1 1.01E+04 4.00 50.3%* (vg/mL) 2 1.62E+04 4.21 2.9% 3 1.63E+04 4.21 7.5% 4 1.65E+04 4.22 1.8% Stool 1 3.23E+02 2.51 2.3% (vg/mg) 2 2.84E+02 2.45 3.3% 3 2.88E+02 2.46 4.6% 4 2.99E+02 2.48 4.2% Urine 1 2.64E+04 4.42 9.5% (vg/mL) 2 2.66E+04 4.42 8.5% 3 2.65E+04 4.42 8.0% 4 2.63E+04 4.42 7.0% SASP1.2 kit was used for extraction and relative high variation was observed in the semen sample with 1 freeze/thaw cycle.
[0284] Long-term −80° C. Storage Stability of Plasma, PBMCs, Saliva, Semen, Urine and Stool—Stability of exemplary Hem-B AAV construct virus in plasma, PBMCs, saliva, semen, stool and urine stored at −80° C. was assessed for up to 6 months. Pooled samples were spiked at 1.0E+05 vg/mL (post 5E+6 cells/mL suspension of PBMCs and 1:10 (w:v) suspension of stool), and three biological replicates per condition were tested at indicated time points. Exemplary Hem-B AAV construct viral genome titer was within ±0.5 LOG of the titer determined at time 0 for up to 6 months. Long-term −80° C. Storage Stability results are summary in
TABLE-US-00050 TABLE 45 Summary of Sample Stability with Long-term −80° C. Storage Sample Type Time Mean Titer Log Mean Titer % CV Plasma 0 wk 6.20E+04 4.79 2.6% (vg/mL) 2 wk 6.34E+04 4.80 7.3% 4 wk 5.68E+04 4.75 8.5% 3 mo 6.81E+04 4.83 19.4% 6 mo 4.71E+04 4.67 3.7% PBMC (vg/μg 0 wk 2.72E+02 2.43 8.4% gDNA) 2 wk 3.98E+02 2.60 5.9% 4 wk 3.85E+02 2.59 0.9% 3 mo 2.73E+02 2.44 14.5% 6 mo 1.85E+02 2.27 7.2% Saliva (vg/mL) 0 wk 3.15E+04 4.50 2.4% 2 wk 3.13E+04 4.50 3.8% 4 wk 2.79E+04 4.45 6.8% 3 mo 3.03E+04 4.48 22.7% 6 mo 2.49E+04 4.40 4.0% Semen (vg/mL) 0 wk 1.74E+04 4.24 3.3% 2 wk 7.86E+03 3.90 43.2%* 4 wk 1.35E+04 4.13 6.5% 3 mo 1.05E+04 4.02 17.1% 6 mo 9.78E+03 3.99 54.0%* Stool (vg/mg) 0 wk 3.12E+02 2.49 1.8% 2 wk 3.12E+02 2.49 3.9% 4 wk 3.06E+02 2.49 0.5% 3 mo 2.86E+02 2.46 8.1% 6 mo 2.45E+02 2.39 1.6% Urine (vg/mL) 0 wk 3.60E+04 4.56 1.1% 2 wk 3.41E+04 4.53 0.9% 4 wk 3.18E+04 4.50 3.1% 3 mo 2.47E+04 4.39 12.8% 6 mo 2.69E+04 4.43 3.0% *SASP1.2 kit was used for extraction and relative high variation was observed in the semen samples at the 2 wk and 6 mo time points
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