TREATMENT OF HYPERBILIRUBINEMIA

Abstract

The invention relates to a nucleic acid sequence useful in the treatment of hyperbilirubinemia, in particular in the treatment of Crigler-Najjar syndrome. More particularly, the nucleic acid sequence of the present invention is a codon-optimized UGT1A1 coding sequence.

Claims

1. An intron which is a modified intron with decreased open reading frames.

2. The intron according to claim 1, which is a modified HBB2 intron, a modified FIX intron, or a modified chicken beta-globin intron.

3. A nucleic acid construct comprising the intron according to claim 1.

4. The nucleic acid construct according to claim 3, further comprising a gene of interest and one or more additional expression control sequences.

5. The nucleic acid construct according to claim 4, wherein the said additional expression control sequence is an ubiquitous or tissue-specific promoter.

6. A vector comprising the intron according to claim 1.

7. The vector according to claim 6, which is a viral vector.

8. The vector according to claim 7, wherein said viral vector is a single-stranded or double-stranded self-complementary AAV vector.

9. The vector according to claim 8, wherein the AAV vector has an AAV-derived capsid.

10. The vector according to claim 9, wherein the AAV vector has an AAV8 capsid.

11. The vector according to claim 9, wherein the AAV vector is a pseudotyped AAV vector.

12. A cell transformed with the nucleic acid construct according to claim 3.

13. A method of gene or cell therapy in a subject comprising expressing a nucleic acid construct according to claim 4 in a subject in need of gene or cell therapy.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0053] FIG. 1 includes graphs showing the levels of messenger RNA observed in Huh-7 cells transfected with plasmid expressing wild-type UGT1A1 or two codon optimized UGT1A1 sequences (panel A) and the quantification by western blot of UGT1A1 protein in the same samples (panel B).

[0054] FIG. 2 includes graphs showing the effect of different intron optimization in the expression of luciferase (panel A) and the effect of HBB2 optimization on UGT1A1 RNA and protein expression level (panel B).

[0055] FIG. 3 is a photograph of a western blot gel showing the expression of UGT1A1 protein from two vectors containing a codon-optimized UGT1A1 coding sequence and containing either the wild-type (UGT1A1 2.0) or an optimized (UGT1A1 2.1) HBB2 intron.

[0056] FIG. 4 is a schematic representation of the in silico analysis of alternate reading frame (ARF) within the wild-type UGT1A1 (A) and a codon-optimized UGT1A1 v2.1 (B) vectors.

[0057] FIG. 5 is a graph showing the levels of total bilirubin (TB) measured every week after the injection of a codon-optimized UGT1A1 vector or of PBS in different rat strains.

[0058] FIG. 6 is a graph showing the levels of total bilirubin (TB) measured every week after the injection of a lower dose of codon-optimized UGT1A1 vector (as compared to the data reported in FIG. 5) or of PBS in different rat strains.

[0059] FIG. 7 includes (A) a graph showing levels of total bilirubin (TB) measured every week after the injection of the three UGT1A1 vectors (as compared to the data reported in FIG. 8); (B) a photograph of a western blot of liver extracts obtained from rats treated with the three vectors and their relative quantification; and (C) a graph presenting the long term evaluation of the efficacy of AAV8-v2.1 UGT1A1 four months after the injection in both male and female animals.

[0060] FIG. 8 is a graph showing the ability of different constructs to correct severe hyperbilirubinemia (Total Bilirubin, expressed as mg/dl) in the mouse model of Crigler-Najjar syndrome. Untreated animals (UNTR) are reported.

[0061] Ranges: Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0062] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.

[0063] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods.

EXAMPLES

[0064] The invention is further described in detail by reference to the following experimental examples and the attached figures. These examples are provided for purposes of illustration only, and are not intended to be limiting.

[0065] Material and Methods

[0066] Codon Optimization and AAV Vector Construct:

[0067] The UGT1A1 underwent codon optimization according to several different algorithms. Additionally, removal of cryptic transcription start sites was implemented throughout the construct. The resulting constructs were either introduced into expression plasmids, or packaged into AAV serotype 8 vectors and tested in vitro and in vivo (rats and mice) for potency.

[0068] The following abbreviations are used throughout this experimental part for these constructs: [0069] WT.0: wild-type UGT1A1 transgene and the wild-type HBB2 intron (SEQ ID NO:5); [0070] WT: wild-type UGT1A1 transgene and the optimized version of the HBB2 intron with some ARFs removed (SEQ ID NO:6); [0071] v2 (or v2.0): comprises codon-optimized UGT1A1 transgene version 2.0 (SEQ ID NO:2) and the wild-type HBB2 intron (SEQ ID NO:5); [0072] v2.1: comprises codon-optimized UGT1A1 transgene version 2.0 (SEQ ID NO:2) and an optimized version of the HBB2 intron with some ARFs removed (SEQ ID NO:6); [0073] v3: comprises codon-optimized UGT1A1 transgene version 3 (SEQ ID NO:3) with an optimized version of the HBB2 intron with some ARFs removed (SEQ ID NO:6); [0074] AAV8-hAAT-wtUGT1A1: AAV8 vector containing the WT construct, under the control of an hAAT promoter wild-type UGT1A1 transgene; [0075] AAV8-hAAT-coUGT1A1v2: AAV8 vector containing the v2 construct, under the control of an hAAT promoter; [0076] AAV8-hAAT-coUGT1A1v2.1: AAV8 vector containing the v2.1 construct, under the control of an hAAT promoter; and [0077] AAV8-hAAT-coUGT1A1v3: AAV8 vector containing the v3 construct, under the control of an hAAT promoter wild-type.

[0078] In Vitro Assay:

[0079] The human hepatocyte cell line Huh7 was transduced at increasing multiplicity of infection (MOI) of 0, 5000 (5), 10000 (10), or 25000 (25) or transfected with the indicated plasmid vectors and lipofectamine. Forty-eight hours after transduction cells were harvested, lysed and microsomal extracts were prepared and loaded on a western blot, where a polyclonal antibody against human UGT1 was used to detect the protein. The constitutively expressed protein calnexin was used as loading control.

[0080] A portion of the cells used for microsomal preparation has been used for mRNA extraction with trizol. Extracted mRNA has been treated with DNAse, retrotranscribed and analyzed by RT-PCR with oligonucleotide primers specific for UGT1A1 sequence. Oligonucleotide primers specific for human serum alkaline phosphatase have been used for the normalization.

[0081] In Silico Analysis:

[0082] The alternative reading frame (ARF) analysis has been performed on the coding strand of the two UGT1A1 sequences with the ORF analysis tool present in the Vector NTI software (Life Technologies). Classic start and stop sites for eukaryotic cells were utilized (respectively ATG as start site and TAA TGA TAG as stop sites). ARFs were considered when their length spans over 50 bp and they have a stop codon in frame with the start.

[0083] Animals:

[0084] Gunn rats, which present a deficiency in the UGT1A1 gene, were injected with vectors at an age of 6-8 weeks. Vectors were delivered via the tail vein in a volume of 0.5 ml. Serum samples were collected weekly to monitor levels of total bilirubin (TB). Untreated affected animals and wild type or healthy littermates were used as controls.

[0085] Ugt1 mutant mice in C57Bl/6 background have been generated previously (Bortolussi et al., 2012). WT littermates were used as a control. Mice were housed and handled according to institutional guidelines, and experimental procedures approved by the local Ethical Committee and the relevant regulatory authorities, with full respect to the EU Directive 2010/63/EU for animal experimentation. The genetic mutation in the Ugt1a gene was transferred to FVB/NJ mouse strain. Animals used in this study were at least 99.8% C57Bl/6 mice or FVB/NJ genetic background, obtained after more than 9 backcrosses with C57Bl/6 mice and FVB/NJ, respectively. Mice were kept in a temperature-controlled environment with 12/12 hours light-dark cycle. They received a standard chow diet and water ad libitum. Vectors were injected intraperitoneally at day 2 (P2) after birth and bilirubin levels were assayed 4 weeks after the injection of the vector.

[0086] Aav Doses:

[0087] The doses of vector administered were indicated in the figure legends.

[0088] Serum Preparation for Rats:

[0089] Blood samples were collected weekly by puncture in retro-orbital sinus, in dry syringes. Blood was centrifuged at 8000 rpm at 4 C., aliquoted and frozen at 20 C.

[0090] Plasma Preparation for Mice:

[0091] Blood samples were collected at 4 weeks after injection in mutant and WT littermates by cardiac puncture in EDTA-collecting syringes. Blood was centrifuged at 2500 rpm, plasma was collected, aliquoted and frozen at 80 C. All procedures were performed in dim light to avoid bilirubin degradation.

[0092] Bilirubin Determination for Rats:

[0093] Total bilirubin determination in serum was performed using Bilirubin Assay Kit (Abnova, ref. KA1614), as described by the manufacturers. We used a volume of 504, of serum to perform the analysis. Absorbance values at 530 nm were obtained by using a multiplate reader (PerkinElmer EnSpire).

[0094] Bilirubin Determination for Mice:

[0095] Total bilirubin determination in plasma was performed using Direct and Total Bilirubin Reagent kit (BQ Kits, San Diego, CA), as described by the manufacturers with minor modifications: the reaction was scaled down and it was performed in a final volume of 300 l (instead of 6000 l), with only 10 l of plasma. Three commercial bilirubin reference standards (Control Serum I, Control Serum II and Bilirubin Calibrator, Diazyme Laboratories, Poway, CA, USA) were included in each set of analysis as quality control. Absorbance values at 560 nm were obtained by using a multiplate reader (Perkin Elmer Envision Plate Reader, Waltham, MA, USA).

[0096] Western Blot on Liver Extracts:

[0097] Snap-frozen liver obtained from the animals injected with either one of the three vectors have been rapidly homogenized. Homogenates have been used for microsome preparation. Microsomal extracts were then loaded on a western blot, where a polyclonal antibody against human UGT1 was used to detect the protein. Protein bands were quantified.

[0098] Results

[0099] Codon-optimized versions of the human UGT1A1 coding sequence were produced and introduced into an expression plasmid. The two optimized UGT1A1 coding sequences (v2 and v3 sequences) and the wild-type sequence have been transfected in Huh-7 cells. Results obtained are reported in FIG. 1. This experiment shows that the two codon optimized sequences are more efficiently translated than the wild-type sequence in human cells in vitro.

[0100] In FIG. 2 panel A are shown luciferase levels produced in Huh-7 cells by transfection with plasmids expressing luciferase under the transcriptional control of the hAAT promoter. Different intronic sequences have been cloned at the 5 of the luciferase coding sequence. Two of them, namely HBB2 and FIX introns, were optimized by removal of ARFs in the sequence done by replacing one nucleotide in ATG codons identified in the wild-type sequence of said introns. The expression of the optimized construct in a hepatic cell line indicates that the removal of ARFs from the intronic sequences increased luciferase expression in vitro in both cases, with the optimized HBB2 intron being particularly potent. In panel B two plasmids were compared, both expressing UGT1A1 under the transcriptional control of the hAAT promoter. V2.0 contains the wild-type HBB2 intron whereas v2.1 contains the optimized version. Data shown indicates that v2.1 plasmid expresses 50% more UGT1A1 than v2.0 without any increase in the mRNA levels.

[0101] Codon-optimized UGT1A1 version 2.0 and 2.1 AAV8 vectors (UGT1A1 2.0 and UGT1A1 2.1, respectively) were tested in vitro. UGT1A1 2.0 and UGT1A1 2.1 vectors differ only by the fact that they contain the wild-type HBB2 intron (SEQ ID NO:5) or a modified HBB2 intron where ARFs have been removed (SEQ ID NO:6), respectively. Results obtained are reported in FIG. 3. This experiment shows that the codon-optimized UGT1A1 vector version 2.1 is more potent than the 2.0 version in human cells in vitro.

[0102] FIG. 4 shows the result of the in silico analysis of alternate reading frame (ARF) within the wild-type UGT1A1 (A) and the codon-optimized UGT1A1v2.1 (B) vectors. The v2.1 vector has only a limited number of ARFs compared to the wild type sequence and mostly in reverse orientation with respect to the promoter. In addition, we can see in FIG. 4 the ARF9 and 10 that are normally present in the HBB2 intron (used in the wild-type UGT1A1 construct represented in A) have been removed from the modified HBB2 intron of SEQ ID NO:6 introduced in the UGT1A1v2.1 optimized vector.

[0103] Then, the codon-optimized AAV8-hAAT-coUGT1A1v2.1 vector was administered at a dose of 510.sup.12 vg/kg. Tail vein injection of the vector has been performed in 6-week-old homozygous Gunn rats (UGT1A1/). In the graph of FIG. 5 are shown the levels of total bilirubin (TB) measured every week, after the injections and in PBS-injected wild type (WT, gray line), heterozygous (UGT1A1+/, dotted line) and homozygous (black line) Gunn rats. All data are expressed as meanSE. Injection of the codon-optimized vector resulted in complete correction of the disease phenotype.

[0104] The AAV8-hAAT-UGT1A1v2.1 vector was also administered at a dose of 510 11 vg/kg. Vector was administered by tail vein injection in 6-week-old homozygous Gunn rats (UGT1A1/). In the graph of FIG. 6 are shown the levels of total bilirubin (TB) measured every week, after the injections and in PBS-injected wild type (WT, gray line), heterozygous (UGT1A1+/, dotted line) and homozygous (black line) Gunn rats. All data are expressed as meanSE. Injection of the codon-optimized vector resulted in complete correction of the disease phenotype.

[0105] The two codon-optimized (v2.1 and v3) and the wild-type AAV8-hAAT-UGT1A1 vectors were further administered at a dose of 510.sup.11 vg/kg. Tail vein injection of the vector has been performed in 6-week-old homozygous Gunn rats (UGT1A1/). In the graph of FIG. 7A are shown the levels of total bilirubin (TB) measured every week, after the injections. All data are expressed as meanSE. As shown in FIG. 7 panel A, the injection of the three vectors resulted in complete correction of the disease phenotype. Two months after the injection, animals were sacrificed and the level of UGT1A1 protein has been quantified by western blot in liver homogenates. In panel B are showed the photographs of western blot with an antibody specific for UGT1A1 protein. The quantification of the bands showed an increase in the quantity of UGT1A1 protein in rats treated with AAV8-hAAT-coUGT1A1v2.1 even if the difference is not significant due to the high variability of the expression levels observed in the different animals.

[0106] Long term efficacy has been evaluated in two month old Gunn rats injected with 510 12 vg/kg of AAV8-v2.1 UGT1A1 vector. Four months after the injection average bilirubin level in blood is 1.75 mg/dL (initial level at DO: 7.49, reduction 77%) in male rats and 0.85 mg/dL (initial level at DO: 6.15 mg/dL, reduction 86%) in female rats. This result, that indicates a long term correction of the phenotype, is particularly striking as compared to a previous study of Pastore et al. reporting a reduction in female rats of only 50% of baseline bilirubin levels using a different construct. Taken together the data showed indicates that the inventive process applied to AAV8-hAAT-coUGT1A1v2.1 resulted in a vector with a better in vivo efficacy as compared to other vectors developed to cure CN.

[0107] We also tested the efficacy of correction of total bilirubin in the mouse model of Crigler-Najjar syndrome. FIG. 8 is a graph showing Total Bilirubin (TB) levels at 1 month post-injection. Animals were injected at day 2 after birth (P2) with a dose of 3E10vg/mouse.

[0108] Untreated affected animals kept alive with 15 days-phototherapy were used as controls (UNTR (PT)).

[0109] This experiment shows that the version 2.1 vector gives the highest level of TB correction of all vectors. All data are expressed as meanSD. Each dot represents a single animal.

REFERENCES

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