Factor IX gene therapy

Abstract

The invention relates to a new, more potent, coagulation Factor IX (FIX) expression cassette for gene therapy of Haemophilia B (HB). Disclosed is a vector for expressing factor IX protein, the vector comprising a promoter, a nucleotide sequence encoding for a functional factor IX protein, and an intron sequence, wherein the intron sequence is positioned between exon 1 and exon 2 of the nucleotide sequence encoding for a functional factor IX protein, and wherein the intron sequence has at least 80% identity to the sequence of SEQ ID NO. 1 as disclosed herein.

Claims

1. A nucleic acid molecule comprising a promoter, wherein the promoter has a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO. 4.

2. The nucleic acid molecule of claim 1, wherein the promoter has a nucleotide sequence having at least 91% identity to the sequence of SEQ ID NO. 4.

3. The nucleic acid molecule of claim 1, wherein the promoter has a nucleotide sequence having at least 92% identity to the sequence of SEQ ID NO. 4.

4. The nucleic acid molecule of claim 1, wherein the promoter has a nucleotide sequence having at least 93% identity to the sequence of SEQ ID NO. 4.

5. The nucleic acid molecule of claim 1, wherein the promoter has a nucleotide sequence having at least 94% identity to the sequence of SEQ ID NO. 4.

6. The nucleic acid molecule of claim 1, wherein the promoter has a nucleotide sequence having at least 95% identity to the sequence of SEQ ID NO. 4.

7. The nucleic acid molecule of claim 1, wherein the promoter has a nucleotide sequence having at least 96% identity to the sequence of SEQ ID NO. 4.

8. The nucleic acid molecule of claim 1, wherein the promoter has a nucleotide sequence having at least 97% identity to the sequence of SEQ ID NO. 4.

9. The nucleic acid molecule of claim 1, wherein the promoter has a nucleotide sequence having at least 98% identity to the sequence of SEQ ID NO. 4.

10. The nucleic acid molecule of claim 1, wherein the promoter has a nucleotide sequence having at least 99% identity to the sequence of SEQ ID NO. 4.

11. The nucleic acid molecule of claim 1, wherein the nucleotide sequence of the promoter is the sequence of SEQ ID NO. 4.

12. The nucleic acid molecule of claim 1, wherein the promoter is: (a) between 330 and 380 nucleotides in length; (b) between 345 and 365 nucleotides in length; or (c) between 350 and 360 nucleotides in length.

13. A host cell comprising the nucleic acid molecule of claim claim 1.

14. A pharmaceutical composition comprising the nucleic acid molecule of claim 1 and one or more pharmaceutically acceptable excipients.

Description

DETAILED DESCRIPTION

(1) The invention will now be described in detail by way of example only with reference to the figures in which:

(2) FIG. 1 shows two vector constructs for delivering factor IX. The top construct HCR hAAT FIX is an existing factor IX gene expression vector used in a gene therapy trial. The bottom construct HCR hAAT FIX TI is the same except that it uses a truncated intron.

(3) FIG. 2 shows FIX expression of the two constructs shown in FIG. 1 in mice following tail vein administration of an identical dose of vector. Expression levels for the HCR hAAT TI FIX vector were 1.8 fold higher than for the HCR hAAT FIX vector which was unexpected based on the prior art.

(4) FIG. 3 shows two factor IX vector constructs. The top construct HCR-hAAT-FIX comprises the wild type factor IX sequence whereas the bottom construct HCR-hAAT-codop-FIX comprises a codon optimised factor IX sequence. In this sequence, exons 1 and 2 have the wild type sequence, whilst exons 3 to 5 have the codon optimised sequence.

(5) FIG. 4 shows FIX expression of the two constructs shown in FIG. 3 in mice following tail vein administration at two doses of vector. Expression levels for the HCR-hAAT-codop-FIX are significantly higher than for the HCR-hAAT-FIX vector.

(6) FIG. 5 shows two further vector constructs for delivering factor IX. The top construct scAAV-LP1-FIXco is a self-complementary vector being used in a haemophilia B clinical trial. The bottom construct scAAV-HLP2-TI-codop-FIX is a single stranded vector which uses a new liver specific promoter (HLP2). In this construct, exons 1 and 2 have the wild type sequence, whilst exons 3 to 5 have the codon optimised sequence.

(7) FIG. 6 shows FIX expression of the two constructs shown in FIG. 5 in mice following tail vein administration of an equivalent number of vector particles as assessed by a gel based titration method. AAV8 capsid pseudotyped single stranded HLP2-TI-codop-FIX mediated at least 3 fold higher levels of FIX when compared to scAAV-LP1-FIXco. This is surprising as self-complementary vectors have previously been shown to mediate substantially higher levels of expression than possible with single-stranded AAV-FIX constructs (Wu et al. Mol Ther. 2008 February; 16(2):280-9 and Nathwani et al. Blood. 2006 Apr. 1; 107(7):2653-61). However, these data show that when optimally configured with regards to inclusion of a strong promoter and efficient splice sites, a single stranded AAV can mediate higher levels of transgene expression than achievable with self-complementary AAV.

(8) The overriding goal of the inventors' research program is to establish a cure for haemophilia B (HB) that is safe, effective and widely available. They established proof-of-concept in a pivotal clinical trial in which a single peripheral vein administration of a self-complementary (sc) adeno-associated viral vector (AAV) expressing a codon optimised FIX transgene (scAAV2/8-LP1-hFIXco) resulted in: (1) stable (>48 months) expression of FIX at 1-6% without long lasting toxicity; (2) discontinuation of prophylaxis in 4/7 participants; (3) reduction in annual bleeding rate of >90% for the 6 subjects in the high dose cohort; and (4) a cost saving so far of £1.5M from reduction in FIX concentrate usage (Nathwani A C et al. N Engl J Med. 365:2357-65, 2011). Obstacles remain to the overriding goal of making AAV-mediated transfer of the normal FIX gene the world-wide curative standard-of-care. Foremost is the body's immune response to cells that have been transduced with the viral vector, resulting in asymptomatic, transient elevation of serum liver enzymes, suggesting local inflammation in the liver. This adverse event only occurred at the high dose but was relatively common (n=4/6). The inventors' efforts have therefore focused on improving potency and transduction efficiency of AAV vectors to enable therapeutic gene transfer in humans with lower, potentially safer vector doses. In pursuit of this goal, the inventors have developed a new more potent FIX expression cassette called HLP2-TI-codop-FIX for AAV mediated gene therapy of haemophilia B.

(9) An initial evaluation compared a single stranded HCR hAAT FIX construct containing a truncated intron 1 (HCR-hAAT-TI-FIX) to an identical construct (HCR-hAAT-FIX) currently being used in an on-going gene therapy trial in mice following tail vein administration of an identical dose of vector. In brief, a dose of 1e11vg was administered into the tail vein of 4-6 week old male C57Bl/6 mice (N=4-6 animals/group). The vector dose was assessed by a gel based titration method described previously (Fagone et al., Hum Gene Ther Methods. 2012 Feb. 23 (1):1-7). FIX levels were assessed using the previously described ELISA method at 4 weeks after gene transfer (Nathwani et al., Mol Ther. 2011 May 19. (5):876-85). A 1.8 fold higher level of FIX in the cohort transduced with HCR hAAT TI FIX was observed per copy of the AAV-FIX transgene (as assessed by a PCR quantification method using primers to hAAT) in the liver at 4 weeks, which was unexpected based on prior art (FIG. 1).

(10) The DNA sequences in HCR-hAAT-FIX were further modified using our in-house codon-optimization algorithm in which codons in the FIX cDNA for a given amino-acid were substituted with the codon most frequently used by the human albumin gene for the same amino-acid since the human albumin is expressed in abundance by the liver. The resulting codop-FIX cDNA was 85% identical to that previously used by our group scAAV-LP1-FIXco (Nathwani et al., Blood. 2006 Apr. 1. 107(7):2653-61). The codop-FIX cDNA was synthesized and cloned downstream of the HCR-hAAT promoter (FIG. 3). To assess the potency of HCR-hAAT-codop-FIX, serotype 8 pseudotyped vector was injected into 4-6 week old male C57Bl/6 mice at a dose of 2e9 or 2e10vg/mouse (N=4-6 animals/dose) based on a gel based titration method. FIX expression in murine plasma was assessed by ELISA at 4 weeks after gene transfer in each dose cohort and compared with the levels achieved in identical dose cohorts transduced with HCR-hAAT-FIX, a vector that contains wild type nucleotide sequence in the FIX cDNA. Codon optimisation of the FIX cDNA resulted in a statistically (one sample t test) significant improvement in transgene gene expression at both dose levels as illustrated in FIG. 4.

(11) Next, the inventors compared the potency of single stranded HLP2-TI-codop-FIX with a self-complementary LP1-FIXco expression cassette currently being used in a haemophilia B clinical trial. In brief, both vectors pseudotyped with serotype 8 capsid were titred using the gel based method to ensure equivalent numbers of self complementary and single stranded AAV particles were administered in 4-8 week old male C57Bl/6 mice. Although transduction with single stranded AAV vectors is limited by the need to convert the single-stranded genome to transcriptionally active double-stranded forms, a head to head comparison showed that for a given vector dose HLP2-TI-codop-FIX mediated at least 3 fold higher levels of FIX in plasma of mice for a given copy of vector in the liver when compared to scAAV-LP1-FIXco (FIG. 6) at 4 weeks after gene transfer, despite the fact that self-complementary vectors are more efficient at forming double stranded transcriptionally active units in the liver.

(12) Sequences

(13) SEQ ID NO. 1—Nucleotide sequence of truncated intron (TI).

(14) SEQ ID NO. 2—Nucleotide sequence of codon optimised FIX. Features: FIX Exon 1: 1-88; FIX Exons 2-5: 89-1386.

(15) SEQ ID NO. 3—Nucleotide sequence of codon optimised FIX containing truncated intron (TI). Features: FIX Exon 1: 1-88; Truncated intron: 89-387; FIX Exon 2-5: 388-1685.

(16) SEQ ID NO. 4—Nucleotide sequence of promoter HLP2.

(17) SEQ ID NO. 5—Nucleotide sequence of HLP2 FIX TI vector. Features: HLP2: 1-354; FIX Exon 1: 425-512; Truncated intron (TI): 513-811; FIX Exons 2-5: 812-2109; bGHpA: 2125-2383.

(18) SEQ ID NO. 6—Nucleotide sequence of codon optimised exons 3 to 5 of FIX.