METHODS AND COMPOSITIONS FOR SUPPRESSING RETROVIRUSES

Abstract

The disclosure provides methods and compositions for suppressing retroviruses, including novel methods for treating a retroviral infection in a human in need of such treatment, comprising delivering a functional meiosis arrest female protein 1 (MARF1) to cells containing a retroviral provirus, together with novel expression constructs comprising a coding sequence encoding a functional MARF1 operatively linked to a promoter, vectors comprising such constructs, and packaging cell lines for use in making such vectors.

Claims

1. A method of treating a retroviral infection in a human in need of such treatment, comprising delivering a functional meiosis arrest female protein 1 (MARF1) to cells containing a retroviral provirus.

2. The method of claim 1, wherein the MARF1 is delivered by means of a DNA vector comprising an expression cassette having a coding sequence encoding a functional MARF1 operatively linked to a promoter, that which will express MARF1 in said cells containing a retroviral provirus.

3. The method of claim 2 wherein the viral vector is a replication-deficient adenoviral (AV) vector or a replication-deficient adeno-associated viral (AAV) vector.

4. The method of claim 1 wherein the retroviral infection is human immunodeficiency virus (HIV).

5. The method of claim 1 wherein the functional meiosis arrest female protein 1 (MARF1) is delivered to the cells containing a retroviral provirus ex vivo, by removing from the patient's body, treating, and returning the cells containing a retroviral provirus.

6. A DNA vector comprising an expression cassette having a coding sequence encoding a functional MARF1 operatively linked to a heterologous promoter.

7. The vector of claim 6 which is a replication-deficient adenoviral (AV) vector or a replication-deficient adeno-associated viral (AAV) vector.

8. A packaging cell line which expresses a vector according to claim 6.

9.-10. (canceled)

Description

DETAILED DESCRIPTION

[0016] The invention this provides, in one embodiment, a method (Method 1) of treating a retroviral infection in a human in need of such treatment, comprising delivering a functional meiosis arrest female protein 1 (MARF1) to cells containing a retroviral provirus. By functional MARF1 is meant a human MARF1 protein or a fragment or variant thereof which retains MARF1 activity in human cells, e.g., which is capable of repressing transposable elements and inhibiting their mobilization.

[0017] For example, the invention provides [0018] 1.1. Method 1, wherein the MARF1 is delivered by means of a DNA vector comprising an expression cassette having a coding sequence encoding a functional MARF1 operatively linked to a promoter, that which will express MARF1 in said cells containing a retroviral provirus. [0019] 1.2. Method 1.1 wherein the DNA vector is a replication-deficient viral vector. [0020] 1.3. Method 1.2 wherein the replication-deficient viral vector is an adenoviral (AV) vector. [0021] 1.4. Method 1.3 wherein the AV vector is not capable of integration into the genome of said cells containing a retroviral provirus. [0022] 1.5. Method 1.2 wherein the replication-deficient viral vector is an adeno-associated viral (AAV) vector. [0023] 1.6. Method 1.5 wherein the AAV vector is not capable of integration into the genome of said cells containing a retroviral provirus. [0024] 1.7. Any of foregoing Methods 1.1 to 1.6 wherein the DNA vector transiently expresses MARF1 in the cells containing a retroviral provirus but does not integrate into the genome of said cells. [0025] 1.8. Any of Methods 1.1, 1.2 or 1.3 wherein the cells containing a retroviral provirus are permanently transformed with a gene constitutively expressing MARF1. [0026] 1.9. Any foregoing method wherein the gene expressing a functional MARF1 comprises a cDNA encoding a human MARF1. [0027] 1.10. Any foregoing method wherein the MARF1 is a human MARF1. [0028] 1.11. Any foregoing method wherein the MARF1 has at least 90% amino acid sequence identity, e.g. at least 95% identity, e.g., at least 99% identity, e.g., as measured by a BLAST algorithm, to a MARF1 selected from GenBank Accession: NP_055462.2, NP_001171927.1 and NP_001171928.1. [0029] 1.12. Any foregoing method wherein the MARF1 has at least 90% amino acid sequence identity, e.g. at least 95% identity, e.g., at least 99% identity, e.g., as measured by a BLAST algorithm, to SEQ ID: 1 [0030] SEQ ID 1:

TABLE-US-00001 1 mmegngtenscsrtrgwlqqdndakpwlwkfsncfsrpeqtlphspqtkeymenkkvave 61 lkdvpsplhagsklfpavplpdirslqqpkiqlssvpkvsccahcpnepstspmrfgggg 121 ggsggtsslihpgalldsqstrtitcqvgsgfafqsasslqnasarnnlagiasdfpsmc 181 lesnlssckhlpccgklhfqschgnvhklhqfpslqgctsagyfpcsdftsgapghleeh 241 isqseltphlctnslhlnvvppvclkgslycedclnkparnsiidaakvwpnipppntqp 301 aplavplcngcgtkgtgkettlllatslgkaaskfgspevavagqvlenlppigvfwdie 361 ncsvpsgrsatavvqrirekffkghreaeficvcdiskenkeviqelnncqvtvahinat 421 aknaaddklrqslrrfanthtapatvvlvstdvnfalelsdlrhrhgfhiilvhknqase 481 allhhanelirfeefisdlpprlplkmpqchtllyvynlpankdgksysnrlrrlsdncg 541 gkvlsitgcsailrfinqdsaeraqkrmenedvfgnriivsftpknrelcetkssnaiad 601 kvkspkklknpklclikdaseqsssakatpgkgsqansgsatkntrwkslqelcrmeskt 661 ghrnsehqqghlrlvvpthgnssaaystpknsgvaepvyktsqkkenlsarsvtsspvek 721 kdkeetvfqvsypsafsklvasrqvspllasqswssrsmspnllnrasplafnianssse 781 adcpdpfangadvqvsnidyrlsrkelqqllqeafarhgkvksvelsphtdyqlkavvqm 841 enlqdaigavnslhrykigskkilvslatgaaskslsllsaetmsvlqdapacclplfkf 901 tdiyekkfghklnvsdlykltdtvaireqgngrlycllpssqarqsplgssqshdgsstn 961 cspiifeeleyhepvcrqhcsnkdfsehefdpdsykipfvilslktfapqvhsllqtheg 1021 tvpllsfpdcyiaefgdlevvqenqggvplehfitcvpgvniataqngikvvkwihnkpp 1081 ppntdpwllrskspvgnpqliqfsrevidllksqpscvipishfipsyhhhfakqcrvsd 1141 ygysklielleavphylqilgmgskrlltlthraqvkrftqdllkllksqaskqvivref 1201 sqayhwcfskdwdvteygvcelidivseipdtticlsqqdnemvicipkrertqdeiert 1261 kqfskdvvdllrhqphfrmpfnkfipsyhhhfgrqcklayygftkllelfeaipdtlqvl 1321 ecgeekiltlteverfkalaaqfvkllrsqkdnclmmtdllteyaktfgytfrlqdydvs 1381 sisaltqklchvvkvadiesgrqiqlinrkslrsltaqllvllmswegtthlsveelkrh 1441 yesthntpinpceygfmtltellkslpylvevftndkmeecvkltslylfaknvrsllht 1501 yhyqqiflhefsmaytkyvgetlqpktyghssveellgaipqvvwikghghkrivvlknd 1561 mksrlsslslspanhenqpsegerilevpeshtaselklgadgsgpshteqellrltdds 1621 pvdllcapvpsclpspqlrpdpvilqsadliqfeerpqepseimilnqeekmeipipgks 1681 ktltsdsssscisaavpvppcpssetsesllskdpvespakkqpknrvklaanfslapit 1741 kl [0031] 1.13. Any foregoing method wherein the MARF1 comprises one or more, or 5 or more, or all of the following conserved sequences from the sequence of SEQ ID NO 1: 352-494, 1487-1562, 1004-1074, 1100-1171, 1260-1330, 1176-1247, 1412-1484, 787-875, 510-582, 1336-1406, 876-937, 795-865. [0032] 1.14. Any foregoing method wherein the MARF1 comprises residues 352-1562 of SEQ ID NO 1. [0033] 1.15. Any foregoing Method 1.1, et seq. wherein the sequence encoding a functional MARF1 is a sequence which encodes a protein which comprises one or more, or 5 or more, or all of the following conserved sequences from the sequence of SEQ ID NO 1: 352-494, 1487-1562, 1004-1074, 1100-1171, 1260-1330, 1176-1247, 1412-1484, 787-875, 510-582, 1336-1406, 876-937, 795-865; e.g., a protein which comprises residues 352-1562 of SEQ ID 1. [0034] 1.16. Any foregoing Method 1.1, et seq. wherein the sequence encoding a functional MARF1 is a sequence which has at least 90% sequence identity, e.g. at least 95% identity, e.g., at least 99% identity, e.g., as measured by a BLAST algorithm, to the coding sequence (CDS) of a nucleotide sequence selected from GenBank Accession: NM_014647.3, NM_001184998.1, and NM_001184999.1. [0035] 1.17. Any foregoing Method 1.1 et seq. wherein the promoter is a viral promoter. [0036] 1.18. Any foregoing Method 1.1 et. seq. wherein the promoter is selected from cytomegalovirus (CMV) and chicken (3-actin (CBA) promoters. [0037] 1.19. Any foregoing Method wherein the retroviral infection is a lentiviral infection. [0038] 1.20. Any foregoing Method wherein the retroviral infection is human immunodeficiency virus (HIV), e.g., HIV-1 or HIV-2, e.g., HIV-1, e.g. HIV-1 Group M, one or more of HIV-1 Group M subtypes A, B or C. [0039] 1.21. Any foregoing method wherein the cells containing a retroviral provirus are white blood cells. [0040] 1.22. Any foregoing method wherein the cells containing a retroviral provirus are CD4+ lymphocytes. [0041] 1.23. Any foregoing Method 1.1 et seq. wherein the functional meiosis arrest female protein 1 (MARF1) is delivered to the cells containing a retroviral provirus [0042] 1.23.1. In vivo, e.g., by injection; or [0043] 1.23.2. Ex vivo, e.g., by removing from the patient's body, treating, and returning the cells containing a retroviral provirus. [0044] 1.24. Any foregoing a method comprising [0045] taking blood from the patient, and optionally further isolating cells containing a retroviral provirus from the blood, e.g. CD4+ cells; [0046] delivering the functional meiosis arrest female protein 1 (MARF1) to the cells containing a retroviral provirus; [0047] and returning said blood or cells to the patent's circulatory system. [0048] 1.25. Any foregoing method wherein the patient receives antiviral medications which will inhibit retroviral expression but will not inhibit AAV vector delivery and expression, e.g., medications selected from one or more of [0049] 1.25.1. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) [0050] 1.25.2. Nucleoside reverse transcriptase inhibitors (NRTIs) [0051] 1.25.3. Protease inhibitors (PIs) [0052] 1.25.4. Fusion inhibitors [0053] 1.25.5. CCR5 antagonists (CCR5s) [0054] 1.25.6. Integrase strand transfer inhibitors (INSTIs) [0055] 1.26. Any foregoing method wherein subsequent to treatment with MARF1, the patient receives antiviral medications to inhibit reinfection by the retrovirus, e.g., medications selected from one or more of [0056] 1.26.1. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) [0057] 1.26.2. Nucleoside reverse transcriptase inhibitors (NRTIs) [0058] 1.26.3. Protease inhibitors (PIs) [0059] 1.26.4. Fusion inhibitors [0060] 1.26.5. CCR5 antagonists (CCR5s) [0061] 1.26.6. Integrase strand transfer inhibitors (INSTIs) [0062] 1.27. Any foregoing method comprising transforming cells containing a retroviral provirus using a vector according to any if Vector 1, et seq. below.

[0063] In another embodiment, the invention provides a DNA vector (Vector 1) comprising an expression cassette having a coding sequence encoding a functional MARF1 operatively linked to a promoter, e.g., a heterologous promoter, e.g. wherein the expression cassette can express said coding sequence to provide MARF1 in cells containing a retroviral provirus. [0064] 1.1. Vector 1 wherein the DNA vector is a replication-deficient viral vector. [0065] 1.2. Vector 1.1 wherein the replication-deficient viral vector is an adenoviral (AV) vector. [0066] 1.3. Vector 1.3 wherein the AV vector is not capable of integration into the genome of said cells containing a retroviral provirus. [0067] 1.4. Vector 1.1 wherein the replication-deficient viral vector is an adeno-associated viral (AAV) vector, e.g. AAV2, 4, 5, 8, or 9, or a synthetic AAV vector such as AAV-DJ (synthetic serotype made from DNA family shuffling of 8 wild type serotypes of AAV, including AAV2, 4, 5, 8, 9, avian, bovine and goat AAV) or self complementary adeno-associated virus (scAAV). [0068] 1.5. Vector 1.4 wherein the AAV vector is not capable of integration into the genome of said cells containing a retroviral provirus. [0069] 1.6. Any foregoing Vector wherein the vector is incapable of integrating to the genome of the cells containing a retroviral provirus but will transiently express MARF1 in said cells, e.g., wherein the vector lacks functional elements necessary for integration, e.g., lacks functional rep and/or cap elements. [0070] 1.7. Any of Vector 1, 1.1, 1.2 or 1.4 wherein the vector is capable of permanently transforming cells containing a retroviral provirus with a gene constitutively expressing a functional MARF1. [0071] 1.8. Any foregoing Vector wherein the MARF1 is a human MARF1. [0072] 1.9. Any foregoing Vector wherein the MARF1 has at least 90% amino acid sequence identity, e.g. at least 95% identity, e.g., at least 99% identity, e.g., as measured by a BLAST algorithm, to a MARF1 selected from GenBank Accession: NP_055462.2, NP_001171927.1 and NP_001171928.1. [0073] 1.10. Any foregoing Vector wherein the functional MARF1 has at least 90% amino acid sequence identity, e.g. at least 95% identity, e.g., at least 99% identity, e.g., as measured by a BLAST algorithm, to SEQ ID: 1. [0074] 1.11. Any foregoing Vector wherein the functional MARF1 comprises one or more, or 5 or more, or all of the following conserved sequences from the sequence of SEQ ID 1: 352-494, 1487-1562, 1004-1074, 1100-1171, 1260-1330, 1176-1247, 1412-1484, 787-875, 510-582, 1336-1406, 876-937, 795-865. [0075] 1.12. Any foregoing Vector wherein the functional MARF1 comprises residues 352-1562 of SEQ ID 1. [0076] 1.13. Any foregoing Vector wherein the sequence encoding a functional MARF1 is a sequence which encodes a protein which comprises one or more, or 5 or more, or all of the following conserved sequences from the sequence of SEQ ID 1: 352-494, 1487-1562, 1004-1074, 1100-1171, 1260-1330, 1176-1247, 1412-1484, 787-875, 510-582, 1336-1406, 876-937, 795-865; e.g., a protein which comprises residues 352-1562 of SEQ ID 1. [0077] 1.14. Any foregoing Vector wherein the sequence encoding a functional MARF1 is a sequence which has at least 90% sequence identity, e.g. at least 95% identity, e.g., at least 99% identity, e.g., as measured by a BLAST algorithm, to the coding sequence (CDS) of a nucleotide sequence selected from GenBank Accession: NM_014647.3, NM_001184998.1, and NM_001184999.1 [0078] 1.15. Any foregoing Vector wherein the promoter is a heterologous promoter, e.g. a promoter different from a natural MARF1 promoter, e.g. wherein the coding sequence encoding a functional MARF1 and the promoter are derived from different species. [0079] 1.16. Any foregoing Vector wherein the promoter is a constitutive promoter. [0080] 1.17. Any foregoing Vector wherein the promoter is a viral promoter. [0081] 1.18. Any foregoing Vector wherein the promoter is selected from cytomegalovirus (CMV) promoter, chicken (3-actin (CBA) promoter, and CAG promoter (i.e., hybrid of the cytomegalovirus (CMV) early enhancer element and chicken beta-actin promoter). [0082] 1.19. Any foregoing Vector further comprising a selectable marker, e.g., an antibiotic resistance gene.

[0083] In other embodiments, the invention provides a packaging cell line which expresses a vector as described in Vector 1 et seq. For example, where the vector is a replication-deficient viral vector, the packaging cell line will express in trans the genes required to permit replication of the vector in the cell line. For example, in some embodiments, to introduce high levels of expressed MARF1 in somatic cells, the vector may be an adenoviral (AV) vector, e.g., a serotype 5 adenovirus, wherein the adenovirus is engineered to be nonreplicative and nonpathogenic, e.g., by deletion of all or part of E1 (which is necessary for replication) and E3 (to provide room for insert of the gene of interest). In this case, the vector can be produced in a packaging cell line that is engineered to express the E1 necessary for the virus to replicate. AV vectors are useful because they infect and express in many cell types including nondividing cells and typically will not integrate into the human genome. In other embodiments, AAV vectors may be used, provided the size of the MARF1 expression cassette is small enough to fit in the AAV vector, e.g., less than about 4.7 Kb. In this embodiment, the recombinant AAV will preferably have the replication and capsid genes are provided in trans (in pRep/Cap plasmid), so that only the two ITRs of AAV genome are left and packaged into virion, while the adenovirus genes required are provided either provided by adenovirus or another plasmid, so that risk for the recombinant AAV to replicate in the cells is very low. For example, in one embodiment, the AAV vector is made by co-transfection of AAV production cells with three plasmids: (1) an AAV2 ITR-containing plasmid carrying the MARF1 expression cassette; (2) a plasmid that carries the AAV2 Rep-Cap; and (3) a plasmid that provides the helper genes isolated from adenovirus. In some embodiments, to avoid risk of triggering an immune response to the vector, the vector is administered ex vivo, to blood cells, for example CD4+ cells, which are removed, transfected, and then returned to the body only after the vector titer is substantially reduced. In some embodiments, the vector will comprise a selectable marker, for example an antibiotic resistance gene, wherein the antibiotic is one such as kanamycin which would kill the packaging cell line in the absence of the resistance gene, so that the packaging cell line is under selective pressure to produce the vector. Non-replicating AV and AAV vectors are commercially available from many sources, e.g., from Vector Biolabs (Malvern, Pa.) or Agilent (Santa Clara, Calif.), for example Agilent's AdEasy adenoviral vector system that uses recombination with a phage produced in E. coli to insert the gene of interest.

[0084] In some embodiments the invention provides MARF1 or a vector comprising a gene for MARF1, e.g., as described in Vector 1, et seq., as a therapeutic agent, e.g., for treatment of a retroviral infection in a human, e.g., in accordance with Method 1 et seq. The invention further provides the use of a sequence expressing MARF1 in the manufacture of a therapeutic agent, e.g., Vector 1 et seq., e.g., for use in treating a retroviral infection in a human, e.g., in accordance with Method 1 et seq.

[0085] Without intending to be bound by theory, it is proposed that the mechanism of MARF1's efficacy to silence a retroviral provirus is similar to the mechanism as seen in MARF1's silencing of retrotransposons in oocytes.

[0086] Certain types of transposable elements, for example Class 1 transposable elements or retrotransposons, are first transcribed from DNA to RNA, then the RNA produced is reverse transcribed to DNA. This copied DNA is then inserted at a new position into the genome. The reverse transcription step is catalyzed by a reverse transcriptase, which may be encoded by a coding region in the transposable element. Retroviruses like HIV and some retrotransposons both contain long terminal repeats (LTRs) and encode reverse transcriptase. Indeed, it is thought that retrotransposons may be descended from ancient retroviral infections. The LTRs act to mediate integration of the retroviral DNA via an LTR specific integrase into the host chromosome. A retroviral provirus can thus be understood as a particular type of eukaryotic retrotransposon, which can produce RNA intermediates that, rather than producing DNA which reintegrates into the host genome, will leave the host cell and produce DNA that can integrate into the genome of other cells.

[0087] While the exact mechanism is not known, it appears that MARF1 recognizes and stimulates methylation of retrotransposons in oocytes. Once the retrotransposons are methylated, their expression is also suppressed in progeny cells. Thus MARF1 is not normally expressed or needed in somatic cells, because the gene silencing of the retrotransposons is already carried out in the oocyte and persists in the somatic cells. But somatic cells are capable of suppressing expression of genes through methylation or other epigenetic means, so the basic mechanism for suppression normally should be available in somatic cells as in oocytes.

[0088] While the regulators for gene expression may vary (primarily MARF1 in oocytes, primarily tumor suppressor factors in somatic cells) the basic machinery for gene suppression (e.g., histone deacetylase and DNA methylase) appears to be ubiquitous, and thus potentially available for use by MARF1 if MARF1 is provided to somatic cells.

[0089] Azidothymidine (AZT) is a nucleoside analog reverse-transcriptase inhibitor that was used as an effective antiviral for HIV until a mutant AZT-resistant strain took over. AZT is shown to have a profound effect on fetal oocyte attrition, increasing oocyte numbers in mice, as FOA is largely dependent on favoring oocytes with less LINE-1 retrotransposon activity. This suggests some functional homology between retroviral proviruses and retrotransposons and supports the theory that a mechanism that silences one could be effective to silence the other.

[0090] One could think of a cell infected with a retrovirus as facing a problem (a potentially disruptive retrotransposon), which is routinely managed with extremely high efficiency at the oocyte level, but which is only rarely seen at the somatic cell level. The proposal involves bringing a tool from the oocyte (MARF1) to fix an analogous problem in a somatic cell.

[0091] In some embodiments, the invention involves administering to HIV positive patients a vector expressing the meiosis arrest female 1 (MARF1) gene, or alternatively, isolating, treating and reintroducing their CD4+ T-cells. The MARF1 should permanently suppress the HIV provirus by selective methylation, so that the provirus will also be suppressed in progeny cells. CD4+ T-cells with HIV provirus suppressed should have a selective advantage over the non-suppressed cells.

[0092] Although the expression of the retroviral provirus in a particular cell line will be permanently silenced following exposure to MARF1, there is still some risk of reinfection from cells that are not treated with the MARF1. This risk can be reduced by continued treatment with conventional antiviral therapies, which suppress infection, or it may be desirable to use a vector that will permanently transform cells containing a retroviral provirus with a gene constitutively expressing MARF1, so that such cells will be resistant to subsequent retroviral infection. This approach has the advantage that the transformed cells will have a selective advantage over the nontransformed cells in the presence of the virus, but may also carry additional risks, however, insofar as the integration of the MARF1 transgene may disrupt other genes, and the long-term effect of constitutive MARF1 expression is not known.

[0093] The foregoing description of certain preferred embodiment(s) and the following examples are in no way intended to limit the invention, its application, or uses.

Example 1Demonstration that MARF1 can Silence Genes in Somatic Cells (CD4+ Cells)

[0094] MARF1 should be able to target first a variety of retrotransposons, and then a retrovirus, as a retrovirus is essentially a retrotranspon with envelope protein. Introducing MARF1 into somatic cells should show a decrease in levels of retrotransposon mRNA and when tested with a retrovirus, show a decrease in virus titer.

[0095] CD4+ cells (SUP-T1, although Jurkat cells expressing CD4 may be used instead) are transformed using adenovirus/retrotransposon hybrid vectors describe by Kubo et al. to insert a L1RP retrotransposon/GFP indicator transgene. See Kubo, S., & Soifer, H. 779. High-Capacity Adenovirus/Retrotransposon Hybrid Vectors for Efficient and Stable Gene Transfer. Molecular Therapy, (2004) 9: S295. AV-MARF1 vector is prepared using Adeno-X Expression System 3 (Clontech) and MARF1 cDNA corresponding to the coding sequence (CDS) of GenBank Accession: NM_014647.3.

[0096] GFP expression is measured in the transformed cells in combination with (i) AV-MARF1 vector or (ii) AV-blank vector (control). Transfection with AV-MARF1 suppresses GFP expression in the cells.

Example 2: Efficacy of MARF1-AV Vector in HIV-Infected Cell Line

[0097] A CD4+ cell line highly susceptible to HIV infection is created, e.g., generally as described in Krowicka H, Robinson J E, Clark R, Hager S, Broyles S, Pincus S H. Use of Tissue Culture Cell Lines to Evaluate HIV Antiviral Resistance. AIDS Research and Human Retroviruses. 2008; 24(7):957-967. The cells are infected with HIV and then one group is treated with MARF1-AV, the other with a blank AV vector. Levels of HIV Gag polyprotein are monitored at different points after infection. Decrease in polyprotein level will correlate with inhibition of replication. Gag protein levels can be determined using a radioimmunoassay (RIA).