GENE THERAPY TREATMENT

Abstract

This disclosure concerns transcription cassettes comprising nucleic acid molecules comprising a nucleotide sequence encoding AP-4 subunits; vectors comprising said transcription cassettes; pharmaceutical compositions comprising said vector; and vectors or compositions for use in the treatment of AP-4-Hereditary Spastic Paraplegia.

Claims

1. An isolated nucleic acid molecule comprising: a transcription cassette comprising a promoter adapted for expression in a mammalian neurone said cassette further comprising a nucleic acid molecule comprising a nucleotide sequence that encodes at least one protein of the AP-4 complex.

2. The isolated nucleic acid molecule according to claim 1, comprising a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: i) a nucleotide sequence, or polymorphic sequence variant, as set forth in SEQ ID NO:1 (AP4B1); ii) a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i); iii) a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the sequence in SEQ ID NO: 1 (AP4B1) wherein said nucleic acid molecule encodes a polypeptide that forms a complex with polypeptides comprising the AP-4 complex; iv) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence as represented in SEQ ID NO: 2 (AP4B1); v) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence wherein said amino acid sequence is modified by addition deletion or substitution of at least one amino acid residue as represented in iv) wherein said polypeptide forms a complex with polypeptides comprising the AP-4 complex.

3. The isolated nucleic acid molecule according to claim 1, comprising a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: i) a nucleotide sequence, or polymorphic sequence variant, as set forth in SEQ ID NO:3 (AP4E1); ii) a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i); iii) a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the sequence in SEQ ID NO: 3 (AP4E1) wherein said nucleic acid molecule encodes a polypeptide that forms a complex with polypeptides comprising the AP-4 complex; iv) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence as represented in SEQ ID NO: 4 (AP4E1); v) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence wherein said amino acid sequence is modified by addition deletion or substitution of at least one amino acid residue as represented in iv) wherein said polypeptide forms a complex with polypeptides comprising the AP-4 complex.

4. The isolated nucleic acid molecule according to claim 1, comprising a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: i) a nucleotide sequence, or polymorphic sequence variant, as set forth in SEQ ID NO: 5 (AP4M1); ii) a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i); iii) a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the sequence in SEQ ID NO: 5 (AP4M1) wherein said nucleic acid molecule encodes a polypeptide that forms a complex with polypeptides comprising the AP-4 complex; iv) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence as represented in SEQ ID NO: 6 (AP4M1); v) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence wherein said amino acid sequence is modified by addition deletion or substitution of at least one amino acid residue as represented in iv) wherein said polypeptide forms a complex with polypeptides comprising the AP-4 complex.

5. The isolated nucleic acid molecule according to claim 1, comprising a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: i) a nucleotide sequence, or polymorphic sequence variant, as set forth in SEQ ID NO: 7 (AP4S1); ii) a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i); iii) a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the sequence in SEQ ID NO: 7 (AP4S1) wherein said nucleic acid molecule encodes a polypeptide that forms a complex with polypeptides comprising the AP-4 complex; iv) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence as represented in SEQ ID NO: 8 (AP4S1); v) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence wherein said amino acid sequence is modified by addition deletion or substitution of at least one amino acid residue as represented in iv) wherein said polypeptide forms a complex with polypeptides comprising the AP-4 complex.

6. The isolated nucleic acid molecule according to any one of claims 1 to 5, wherein said cassette is adapted for expression in a motor neurone.

7. The isolated nucleic acid molecule according to claim 6 wherein said promoter is selected from the group consisting of: chicken beta actin (CBA) promoter, chicken beta actin hybrid (CBh) promoter, CAG promoter, neuronal and glial specific promoters including synapsin 1, Hb9, MeP229 and GFAP promoter sequences, as well as AP-4 subunit specific promoter regions including AP4B1, AP4E1, AP4M1 and AP4S1.

8. The isolated nucleic acid molecule according to claim 7 wherein said promoter sequence comprises a nucleic acid molecule comprising a nucleotide sequence as set forth in SEQ ID NO: 27, or a nucleotide sequence that is a polymorphic sequence variant of SEQ ID NO: 27.

9. An expression vector comprising an isolated nucleic acid molecule according to any one of claims 1 to 8.

10. The expression vector according to claim 9, wherein said expression vector is a viral based expression vector.

11. The expression vector according to claim 10 wherein said viral based vector is AAV9.

12. The expression vector according to claim 10 wherein said viral based vector is a lentiviral vector.

13. The expression vector according to claim 9 wherein said viral based vector comprises the nucleotide sequence set forth in SEQ ID NO: 19 (AP4B1).

14. The expression vector according to claim 9 wherein said viral based vector comprises the nucleotide sequence set forth in SEQ ID NO: 20 (AP4B1).

15. The expression vector according to claim 9 wherein said viral based vector comprises the nucleotide sequence set forth in SEQ ID NO: 21 (AP4S1).

16. The expression vector according to claim 9 wherein said viral based vector comprises the nucleotide sequence set forth in SEQ ID NO: 22 (AP4S1).

17. The expression vector according to claim 9 wherein said viral based vector comprises the nucleotide sequence set forth in SEQ ID NO: 23 (AP4E1).

18. The expression vector according to claim 9 wherein said viral based vector comprises the nucleotide sequence set forth in SEQ ID NO: 24 (AP4E1).

19. The expression vector according to any one of claims 9 to 12—wherein said viral based vector comprises further the nucleotide sequence set forth in SEQ ID NO: 25 or 26.

20. A pharmaceutical composition comprising an expression vector according to any one of claims 9 to 19 and an excipient or carrier.

21. An expression vector according to any one of claims 9 to 19 for use as a medicament.

22. An expression vector according to any one of claims 9 to 19 for use in the treatment of AP-4-Hereditary Spastic Paraplegia (AP-4-HSP).

23. The expression vector according to the use of claim 22 wherein the AP-4-HSP is SPG47, SPG50, SPG51 or SPG52.

24. A cell transfected with an expression vector according to any one of claims 9 to 19.

25. The cell according to claim 24 wherein said cell is a neurone.

26. The cell according to claim 25 wherein said cell is a motor neurone.

27. A method to treat or prevent AP-4 Hereditary Spastic Paraplegias (HSP) comprising administering a therapeutically effective amount of an expression vector according to any one of claims 9 to 19 to prevent and/or treat AP-4-HSP.

28. The method according to claim 27 wherein said AP-4-HSP is SPG47, SPG50, SPG51 or SPG52.

29. A diagnostic method to genotype a subject to determine whether the subject has a mutation in one or more AP-4 gene sequences comprising the steps: i) obtaining a biological sample from a subject to be tested and extracting nucleic acid from said biological sample; ii) sequencing the nucleic acid to obtain a nucleotide sequence of AP4B1, AP4E1, AP4M1 and AP4S1 in said subject; iii) comparing the obtained genomic sequence with a normal matched control nucleotide sequence to identify nucleotide sequence differences; and iv) determining whether the test sample is modified in an AP-4 gene sequence and whether said modification is associated with AP-4-HSP.

30. The diagnostic method according to claim 29, wherein said method further comprises the administration of at least one expression vector according to any one of claims 9 to 19 to prevent or treat AP-4-HSP.

31. The diagnostic method according to claim 29 or 30, wherein said AP-4-HSP is selected from the group consisting of: SPG47, SPG50, SPG51 and SPG52.

32. The diagnostic method according to any one of claims 29 to 31, wherein said genomic sequence comprises SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, or a polymorphic sequence variant thereof.

33. A transcription cassette comprising: a first nucleic acid molecule comprising a nucleotide sequence as set forth in SEQ ID NO: 27, or a nucleotide sequence that is a polymorphic sequence variant of SEQ ID NO: 27, wherein said nucleic acid molecule is a transcription promoter and operably linked to a second nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide wherein the first nucleic acid molecule regulates transcription of the second nucleic acid molecule.

34. The transcription cassette according to claim 33 wherein said first nucleic acid molecule comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 27.

35. The transcription cassette according to claim 33 or 34 wherein said second nucleic acid molecule comprises a nucleotide sequence that encodes at least one polypeptide of the AP-4 complex.

36. The transcription cassette according to claim 35 wherein said second nucleic acid molecule comprises or consists of a nucleotide sequence as represented in SEQ ID NO: 1, or polymorphic sequence variant thereof.

37. The transcription cassette according to claim 35 wherein said nucleic acid molecule comprises or consists of a nucleotide sequence as represented in SEQ ID NO: 3, or polymorphic sequence variant thereof.

38. The transcription cassette according to claim 35 wherein said nucleic acid molecule comprises or consists of a nucleotide sequence as represented in SEQ ID NO: 5, or polymorphic sequence variant thereof.

39. The transcription cassette according to claim 35 wherein said nucleic acid molecule comprises or consists of a nucleotide sequence as represented in SEQ ID NO: 7, or polymorphic sequence variant thereof.

40. An expression vector comprising a transcription cassette according to any one of claims 33 to 39.

41. The expression vector according to claim 40, wherein said expression vector is a viral based expression vector.

42. The expression vector according to claim 41 wherein said viral based vector is AAV9.

43. The expression vector according to claim 41 wherein said viral based vector is a lentiviral vector.

Description

EXAMPLE 1

[0167] The size of the human AP4B1 cDNA open reading frame (2,800 bp) means that a simple gene replacement option is technically feasible and amenable to typical viral delivery approaches such as using a single-stranded adeno-associated virus (AAV) which has an insertion limit of 4,000 bp. We designed AAV vectors to achieve the strongest level of transgene expression (FIG. 2A): 1) An expression cassette was developed involving the 0.8 kb CBh promoter and 130 bp SV40 polyA to drive expression of the human AP4B1. The CBh promoter has been reported to mediate efficient transgene expression in rodents and non-human primates; 2) A vector expressing an N-terminal V5 viral epitope-tagged human AP4B1 cDNA allowing in vitro and in vivo detection of AP4B1 restoration in the absence of suitable anti-AP4B1 antibodies; 3) A V5-tagged AP4B1 construct expressed from a lentiviral vector enabling in vitro validation of efficacy in cell types that are not efficiently transduced by AAV9 (e.g. fibroblasts). All constructs have been shown to efficiently express their viral cargos upon transfection or transduction in HeLa cells (FIG. 2B,C), primary rat cortical neurons (FIG. 2D), and human fibroblasts (FIG. 2E) as determined by western blotting, RT-qPCR and immunocytochemistry. The viral constructs efficiently restore expression of the AP4B1 protein in both a CRISPR generated AP4B1-knockout HeLa cell line and fibroblasts from SPG47 patients lacking endogenous AP4B1 (FIG. 2 B,C,E). Expression of V5-tagged AP4B1 in SPG47 patient fibroblasts also rescues both overexpression and mis localisation of ATG9A (FIG. 3).

EXAMPLE 2

[0168] Intrathecal delivery of AAV9 viral vectors (AAV9-AP4B1, AAV9-V5-tagged-AP4B1 or AAV9-GFP) via the cisterna magna in wildtype C57BL6/J mice, resulted in widespread transduction of multiple tissues including the brain (FIG. 4A,B), a route of delivery known to lead to efficient gene transfer to CNS as described in our previous work. Long-term pilot safety studies in wild type mice treated with AAV9-AP4B1 show no apparent side effects on body weight, motor function or clinical observations up to the age of 6 months (FIG. 4C-E).

EXAMPLE 3

[0169] In tandem with the development of the therapeutic viral vector, generation of CRISPR-based Ap4b1-knockout (EM5 Ap4b1−/−) mouse line was outsourced to the Jackson Laboratory then transferred to SITraN in Sheffield for full characterisation. Phenotypic characterisation of the line revealed that EM5 Ap4b1−/− lines show consistent progressive motor deficits as demonstrated by roratod, open field and CatWalk footprint testing (FIG. 5A-C) and clasping (FIG. 6). Preliminary MRI analysis revealed thin corpus callosum in of EM5 Ap4b1−/− when compared to wild type mice (FIG. 7A,B), a clinically relevant phenotype since the same pathological phenotype has been reported in SPG47 patients. This observation has been confirmed by H & E staining (FIG. 7C,D). Further analysis of MRI study is ongoing to assess other readouts such as the overall size of lateral ventricles, and alterations in white matter volume, phenotypes already reported in AP4E1−/− and human AP4 cases.

[0170] A mouse model of SPG47 was generated by Jackson labs (Bar Harbor, Me.): The C57BL/6J-Ap4b1.sup.em5Lutzy/J model (Stock #031349) contains a mutant Ap4b1 gene with a 76 bp deletion within exon 1. The C57BLJ6J-Ap4b1.sup.em4Lutzy/J model (Stock #031062) contains a 78 bp deletion+1 bp deletion in exon 1 of mouse Ap4b1 gene. Neither is known to be a human pathogenic mutation but are predicted to generate frameshift that result in early nonsense mutations. The strain was developed with CRISPR/Cas 9 technology and a mutagenic oligonucleotide. Plasmids encoding a signal guide RNA designed to introduce a 76 bp deletion within exon 1 of the Ap4b1 gene and the cas9 nuclease were introduced into the cytoplasm C57BL/6J-derived fertilized eggs with well recognized pronuclei. Correctly targeted embryos were transferred to pseudopregnant females. Correctly targeted pups were identified by sequencing and PCR and further bred to C57BL/6J to develop the colony. PCR genotyping allows identification of wildtype, heterozygotes and homozygous knockout mice.

[0171] Further characterization of the model was executed by Azzouz lab of University of Sheffield. RT-PCR and qRT-PCR showed very low levels of Ap4b1 mRNA in the mutant mice consistent with nonsense mediate decay. Western blots showed the absence of a cross reacting band of ˜85 kDa normally expressed at various levels in brain, muscle, spinal cord, liver and heart of wildtype mice.

[0172] The em5lutzy line has been examined in more detail. Weight gain in wildtype and Ap4b1−/− female mice is similar while weight gain in male Ap4b1−/− mice is slower than male wildtype mice with a significantly lower weight at >6 months. A number of behavioral assessments were conducted including gait analysis clasping, open field testing and rotarod performance (FIGS. 5 and 6). All of these showed a deficiency in the Ap4b1−/− mice. Gait analysis showed paw angle for hind limbs was abnormally wide for mutant mice compared to wildtype (FIG. 5D). In the clasping assay mutant mice had a higher percent clasping at all ages compared with wildtype (FIG. 6B). Open field activity was comparable in wildtype and mutant mice at 6 months of age but there was age-dependent decrease in wildtype mice that was absent in mutant mice (FIG. 5B). The rotarod showed the most consistent difference with latency to fall on rotarod is ˜15% lower at all ages tested form 50 to 120 days of age (FIG. 5A). Further characterization is underway by open field activity monitoring, catwalk, hind limb clasping, MRI, histopathology, ATG9A localization and protein levels. Motor deficiency is a key feature of the clinical presentation of SPG47/AP4B1 deficiency and therefore these mouse phenotypes are directly relevant to the human disease and appropriate markers for assessing potential therapeutics.

[0173] Further morphological and histopathological studies on these Ap4b1−/− knockout mice are ongoing. A published study on a knockout mouse for the Ap4e1 gene shows a thin corpus callosum and axonal swellings in various areas of the brain and spinal cord. Immunohistochemical analyses showed that the transmembrane autophagy-related protein 9A (ATG9A) is more concentrated in the trans-Golgi network (TGN) and depleted from the peripheral cytoplasm both in various neuronal types in Ap4e1 knockout mice. This leads to distal axonal swellings containing accumulated ER, defective autophagosomes, and shortening of the axons observable both in vitro and in vivo.

Proof of Concept. Treatment of SPG47 Mouse Model by Intra-Cisterna Magna AAV9

[0174] Three studies have been conducted to assess the impact of intra-cisterna magna AAV9-hAP4B1 in mouse C57BL/6J-Ap4b1.sup.em5Lutzy/J model using juvenile of neonatal mice.

Cisterna Magna Injection of p1 Mice.

[0175] Proof of concept was demonstrated in newborn Ap4b1−/− mice. By using neonates, it was assumed that there would be maximum benefit to relieve any developmental manifestations of disease. Experimental design (1) included 4 cohorts of homozygous knockout mice with either of two doses of the AAV9-HAP4B1 vector expressing human cDNA for AP4B1; empty vector expressing epitope tag (AAV9-V5) or untreated. Positive control was untreated wildtype mice.

[0176] Stocks of AAV9 viral vectors were produced by transfecting adherent human embryonic kidney HEK293T cells and purification of the vector using iodixanol gradient centrifugation. Briefly, HEK293T cells were transfected with packaging plasmids pHelper (Stratagene; Stockport, UK), pAAV2/9 (kindly provided by J. Wilson, University of Pennsylvania) and one of the transgene plasmids (e.g. AAV9-CBh-AP4B1) at 2:1:1 ratio, respectively, using polyethylenimine (1 mg/ml) in serum-free Dulbecco's modified Eagle's medium. At 3 days post-transfection, supernatant containing cell-released virus was harvested, treated with benzonase (10 unit/ml; Sigma, Poole, UK) for 2 hours at 37° C. and concentrated to equal to approximately 24 ml using Amicon Ultra-15 Centrifugal 100K Filters (Millipore, Watford, UK). Iodixanol gradient containing 15, 25, 40, and 54% iodixanol solution in phosphate-buffered saline (PBS)/1 mmol/1 MgCl2/2.5 mmol/l KCl and virus solution was loaded and centrifuged at 69,000 revolutions per minute for 90 minutes at 18° C. After ultracentifugation, the virus fractions were visualized on a 10% polyacrylamide gel, stained using SYPRO Ruby (Life Technologies, Paisley, UK) according to the manufacturer's guidelines. The highest purity fractions (identified by the presence of the three bands corresponding to VP1, VP2, and VP3) were pooled and concentrated further in the final formulation buffer consisting of PBS supplemented with an additional 35 mmol/l NaCl using Amicon Ultra-15 Centrifugal 100K filters. Viral titers were determined by quantitative PCR assays.

TABLE-US-00009 TABLE 1 Study design for P1 mouse efficacy experiment.sup.1 N (Male/ Total Cohort Genotype Treatment/dose Female) N 1 Ap41b1−/− AAV9-hAP4B1 (5 × 10.sup.13 gc/kg) 6/4 10 2 Ap41b1−/− AAV9-hAP4B1 (2 × 10.sup.13 gc/kg) 8/1 9 3 Ap41b1−/− AAV9-V5 (5 × 10.sup.13 gc/kg) 5/4 9 4 Wildtype Untreated 9/8 17 5 Ap41b1−/− Untreated 1/6 7 .sup.1Assessments included general observation, weight and accelerating rotarod every 3 weeks, open field, clasping and catwalk testing at 3-month intervals.

[0177] There was no mortality observed for any mice in any of these cohorts. Weigh gain was assessed every 2 weeks (FIG. 9) with a clear pattern of slower weight gain by untreated Ap4b1−/− mice of either gender compared to wildtype controls. Neonatal injection of high dose AAV9-hAP4B1 restored male weight gain to levels in wildtype mice whereas it had no impact on weight gain in female mice.

[0178] Phenotype was assessed by the clasping assay in which mice with certain neurological defects (but not wildtype mice) clasp their limbs when suspended by the tail. The data (FIG. 10) clearly show an age-dependent increase with 83% of knockout mice showing clasping response compared to 7% wildtype controls at 9-month age (p<0.001; Chi square test). Treatment of knockout mice by a high dose of AAV9-hAP4B1 vector significantly mitigated clasping response at 9 months (p<0.01) although response did not achieve the level in wildtype mice (p<0.0001). By contrast, control vector AAV9-CBH-V5, had no impact on clasping response. Also, use of low dose AAV9-hAP4B1 showed no impact with age dependent clasping comparable to that of untreated Ap4b1−/− mice suggesting that the target dose is at least 5×10.sup.13 gc/kg.

[0179] Phenotype was also assessed by accelerating rotarod (FIG. 11) at 4-week intervals. As with weight gain, the data are more clearly understood when male and female mice are considered separately. For male mice there was clear improvement in performance of high dose AAV9-hAP4B1 treated knockouts up to a level comparable to that of wildtype mice of the same age. For female mice, the knockouts did not show a defect in rotarod performance and therefore there is no possibility of seeing any response to AAV-hAP4B1 delivery into cistern magna. We note that substantial differences in performance between males and female mice are known in both wildtype [12] and mouse models of neurological diseases including GNAO1 [13], ALS [14, 15] and Alzheimer's [16].

[0180] Performance was also assessed by overall activity in open field testing in a subset of mice. By this measure there was a clear difference between wildtype and knockout mice but no impact of treatment on activity. A similar pattern was seen in both male and female cohorts.

[0181] Status of the AP4 complex was assessed by measuring the AP4E1 levels (FIG. 12). An important observation is the depletion of AP4E1 unit of the AP4 complex in AP4b1.sup.−/− when comparison to WT mice. The same observation has been reported in cell model systems of

[0182] SPG47 including human cells isolated from AP4 patients. AAV9-AP4B1 gene transfer increase AP4E1 levels in the AP4B1−/− mouse spinal cord (FIG. 12A) and Heart (FIG. 12B) suggesting restoration of AP4 complex (FIG. 13).

CM delivery in P1 wild type mice: Pilot Safety Study

[0183] This aim of this pilot study was to assess the biodistribution, stability of viral-mediated transgene expression and potential adverse effects of AAV9-hAP4B1 in wildtype mice (FIG. 4). One viral vector will be used, expressing a full-length copy of the human AP4B1 gene containing an N-terminal V5 viral epitope tag. The viral vectors were delivered directly into the cisterna magna of P1/2 pups, using stereotaxic apparatus containing a 33-gauge Hamilton syringe with automated perfusion pump. Five p of solution was administered at 1 uL per minute. Biodistribution of the virus, expression, body weight and motor function (rotarod) were assessed at 4 weeks and 6 months post injection.

TABLE-US-00010 TABLE 8 Sequence summary SEQ ID NO Description 1 Homo sapiens adaptor related protein complex 4 subunit beta 1 (AP4B1)-DNA 2 AP4B1-Amino acid sequence 3 Homo sapiens adaptor related protein complex 4 subunit epsilon 1 (AP4E1)-DNA 4 AP4E1-Amino acid sequence 5 Homo sapiens adaptor related protein complex 4 subunit sigma 1 (AP4M1)-DNA 6 AP4M1-Amino acid sequence 7 Homo sapiens adaptor related protein complex 4 subunit mu 1 (AP4S1)-DNA 8 AP4S1-Amino acid sequence 9 CRISPR-sequence 10-18 Primer sequences 19 hSYN1-hAP4B1 20 CBh-hAP4B1 21 SYN 1hAP4S1-3'UTR 22 MeP229hAP4S1 23 MeP229hAP4E1 24 SYN1hAP4E1 25 Stuffer sequence 1 26 Stuffer sequence 2 27 AP4 promoter 28 AP4 promoter forward primer 29 AP4 promoter reverse primer 30 Mininmal promoter sequence

REFERENCES

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