US20220331409A1 - Factor ix gene therapy - Google Patents
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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.
Description
- This application is a continuation of U.S. patent application Ser. No. 16/563,193, filed on Sep. 6, 2019, which is a continuation of U.S. patent application Ser. No. 15/525,836, filed May 10, 2017, which is a national phase entry under 35 U.S.C. § 371 of International Patent Application PCT/GB2015/053438, filed Nov. 12, 2015, designating the United States of America and published in English as International Patent Publication WO/2016/075473 on May 19, 2016, which claims the benefit under Article 8 of the Patent Cooperation Treaty to United Kingdom Patent Application Serial No. 1420139.6, filed Nov. 12, 2014, the entirety of each of which are incorporated herein by this reference.
- The invention relates to a new more potent coagulation factor IX (FIX) expression cassette for gene therapy of haemophilia B (HB).
- Pursuant to 37 C.F.R. § 1.821(c) or (e), files containing a TXT version of the Sequence Listing have been submitted concomitant with this application, the contents of which are hereby incorporated by reference.
- Haemophilia B, an X-linked life threatening bleeding disorder affects 1:30,000 males. Current treatment involves frequent intravenous injections (2-3 times per week) of FIX protein. This treatment is highly effective at arresting bleeding but it is not curative and is extremely expensive (£150,000/patient/year), thus making it unaffordable by the majority of haemophilia B patients in the World. Gene therapy for HB offers the potential for a cure through persistent, endogenous production of FIX following the transfer of a normal copy of the FIX gene to an affected patient. Even a small rise in circulating FIX to >1% of normal levels can significantly ameliorate the severe bleeding phenotype.
- The inventors have recently successfully piloted a gene therapy approach using adeno-associated viral vectors (AAV) to mediate transfer and expression of the gene for normal coagulation factor IX (FIX) in the liver. Preliminary results of this successful academic, investigator led, trial were published in the New England Journal of Medicine, with experts in the field lauding the work, and calling it a “landmark study”.
- Despite the initial success, some obstacles remain to the overriding goal of making AAV-mediated transfer of the normal FIX gene the world-wide curative standard-of-care. Foremost among these is the body's immune response to cells that have been transduced with the viral vector that resulted in asymptomatic, transient elevation of serum liver enzymes, suggesting local inflammation in the liver. Treatment with a short course of steroids led to rapid normalisation of the liver enzymes and continued expression of FIX, but at levels below those that had been generated before the episodes of liver inflammation. Since this observation only occurred at the high dose level, current efforts are focused on improving the potency and transduction efficiency of AAV vectors so that therapeutic gene transfer can be achieved with lower, potentially safer vector doses. To this end, the inventors have developed a new AAV expression cassette, referred to as HLP2-TI-codop-FIX, which mediates transgene expression at levels that are up to four fold higher than previously observed with the self-complementary (sc) LP1-hFIXco cassette used in the previous clinical trial. HLP2-TI-codop-FIX improves the safety profile of AAV mediated gene transfer whilst reducing the burden on vector production.
- The aspects of HLP2-TI-codop-FIX which make it different from previously used vectors include the following:
- 1. It is a single stranded vector. This allows a larger transgene to be packaged in the AAV than with self-complementary AAV;
- 2. A new synthetic liver promoter (HLP2). This promoter is a modified promoter which contains an extra enhancer region to increase expression;
- 3. Truncated 299 bp intron (TI) derived by engineering sequences from wild
type FIX intron 1A which spans 6.2 kb. The truncated intron (TI) in this expression cassette is placed in its native position between exon 1 andexon 2 of the FIX. Despite its smaller size, it increases expression levels by 1.6 fold over that observed with wild typefull length intron 1A; and - 4. New codon optimised FIX to increase expression over wild-type sequence by almost two fold (
FIG. 2 ). - In a first aspect of the invention, there is provided 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. - The intron sequence in the vector is derived from
intron 1A of the wild type FIX gene. It has been found that truncating the sequence ofintron 1A causes expression of the vector to be increased. It is thought that the truncation ofintron 1A may delete a repressor element in the intron. - Truncation of the
intron 1A sequence also results in the nucleotide sequence of the vector being shorter which allows more efficient packaging of the vector in a viral delivery system. - In the vector of the invention, the intron sequence is positioned between exon 1 and
exon 2 of the nucleotide sequence encoding for a functional factor IX protein. The complete sequence of the human FIX gene is well known, including the sequences of the introns and exons. For example, this information can be found on Genbank (http://www.ncbi.nlm.nih.gov/genbank) under accession numbers J00137.1; B C109215. 1; BC109214.1; AB186358.1; and FR846239.1. Therefore, it is well within the abilities of a skilled person to determine where in the FIX coding sequence the intron sequence is located and in particular, betweenexon 1 and 2 of the FIX gene. Generally, when protein coding sequences are incorporated into vectors, the coding sequence does not contain any introns. - The intron sequence has at least 80% identity to the sequence of SEQ ID NO. 1. In some embodiments, the intron sequence has at least 82% identity to the sequence of SEQ ID NO. 1. In other embodiments, the intron sequence has at least 84% identity to the sequence of SEQ ID NO. 1. In particular embodiments, the intron sequence has at least 86% identity to the sequence of SEQ ID NO. 1. In further embodiments, the intron sequence has at least 88% identity to the sequence of SEQ ID NO. 1. In some embodiments, the intron sequence has at least 90% identity to the sequence of SEQ ID NO. 1. In other embodiments, the intron sequence has at least 91% identity to the sequence of SEQ ID NO. 1. In particular embodiments, the intron sequence has at least 92% identity to the sequence of SEQ ID NO. 1. In further embodiments, the intron sequence has at least 93% identity to the sequence of SEQ ID NO. 1. In some embodiments, the intron sequence has at least 94% identity to the sequence of SEQ ID NO. 1. In other embodiments, the intron sequence has at least 95% identity to the sequence of SEQ ID NO. 1. In particular embodiments, the intron sequence has at least 96% identity to the sequence of SEQ ID NO. 1. In further embodiments, the intron sequence has at least 97% identity to the sequence of SEQ ID NO. 1. In some embodiments, the intron sequence has at least 98% identity to the sequence of SEQ ID NO. 1. In other embodiments, the intron sequence has at least 99% identity to the sequence of SEQ ID NO. 1. In particular embodiments, the intron sequence has the sequence of SEQ ID NO. 1.
- The truncated intron sequence is preferably between 270 and 330 nucleotides in length. In some embodiments, the intron sequence is between 280 and 320 nucleotides in length. In other embodiments, the intron sequence is between 290 and 310 nucleotides in length. In particular embodiments, the intron sequence is between 295 and 305 nucleotides in length. In specific embodiments, the intron sequence is between 295 and 300 nucleotides in length.
- The vector contains a nucleotide sequence encoding for a functional factor IX protein so that when this sequence is expressed, a functional FIX protein is produced by the cell in which the vector is contained. When expressed in a subject, e.g. a human patient, the functional FIX protein is one which can take part in the coagulation cascade to allow blood dotting to take place. This causes a decrease in the time it takes for blood to clot in a subject suffering from haemophilia B. The functional FIX protein can be activated to produce the enzymatically active factor IXa which can convert factor X to factor Xa.
- The sequence of the FIX protein produced by expression of the vector may be the wild type FIX sequence. In one embodiment, the nucleotide sequence encoding for a functional factor IX protein consists of exons 1 to 5 of the FIX gene. The FIX gene normally contains 8 exons, with exons 6 to 8 encoding an untranslated region. The advantage of using a shorter sequence is that it can be incorporated into a vector more easily and more effectively.
- As mentioned above, the sequence of the FIX gene is well known to a skilled person and therefore, it would be well within the capabilities of a skilled person to produce a nucleotide sequence encoding for a functional factor IX protein.
- The nucleotide sequence encoding for a functional FIX protein preferably has at least 80% identity to the sequence of SEQ ID NO. 2. In some embodiments, the nucleotide sequence has at least 82% identity to the sequence of SEQ ID NO. 2. In other embodiments, the nucleotide sequence has at least 84% identity to the sequence of SEQ ID NO. 2. In further embodiments, the nucleotide sequence has at least 86% identity to the sequence of SEQ ID NO. 2. In particular embodiments, the nucleotide sequence has at least 88% identity to the sequence of SEQ ID NO. 2. In some embodiments, the nucleotide sequence has at least 90% identity to the sequence of SEQ ID NO. 2. In other embodiments, the nucleotide sequence has at least 91% identity to the sequence of SEQ ID NO. 2. In further embodiments, the nucleotide sequence has at least 92% identity to the sequence of SEQ ID NO. 2. In particular embodiments, the nucleotide sequence has at least 93% identity to the sequence of SEQ ID NO. 2. In some embodiments, the nucleotide sequence has at least 94% identity to the sequence of SEQ ID NO. 2. In other embodiments, the nucleotide sequence has at least 95% identity to the sequence of SEQ ID NO. 2. In further embodiments, the nucleotide sequence has at least 96% identity to the sequence of SEQ ID NO. 2. In particular embodiments, the nucleotide sequence has at least 97% identity to the sequence of SEQ ID NO. 2. In some embodiments, the nucleotide sequence has at least 98% identity to the sequence of SEQ ID NO. 2. In other embodiments, the nucleotide sequence has at least 99% identity to the sequence of SEQ ID NO. 2. In preferred embodiments, the nucleotide sequence has the sequence of SEQ ID NO. 2.
- When the nucleotide sequence encoding for a functional FIX protein has sequence identity to the sequence of SEQ ID NO. 2, this does not include the intron sequence which is positioned between exon 1 and
exon 2 of the nucleotide sequence encoding for a functional factor IX protein. For example, when the nucleotide sequence encoding for a functional FIX protein has the sequence of SEQ ID NO. 2 and the intron sequence has the sequence of SEQ ID NO. 1, in the nucleotide sequence of the actual vector, the sequence of SEQ ID NO. 1 will appear within SEQ ID NO. 2. This means that there will be a portion of SEQ ID NO. 2 followed by SEQ ID NO. 1 followed by the remaining portion of SEQ ID NO. 2. - The sequence of SEQ ID NO. 2 is a codon optimised FIX nucleotide sequence in which the sequence of exons 3 to 5 has been codon optimised. The sequence of
exons 1 and 2 of SEQ ID NO. 2 is the wild type FIX sequence. The sequence of exons 3 to 5 has not been codon optimised in a normal way. Instead, the codons have been selected based on the codons used for proteins which are expressed at a high level in the liver. The reason for this is that the vector is normally expressed in the liver. This special codon optimisation process has been found to produce a nucleotide sequence which gives surprisingly high expression. The wild type sequence has been used forexons 1 and 2 to help to ensure that splicing is not affected when the intron is removed during processing of the RNA molecule expressed from the nucleotide sequence. - As described above, the vector comprises 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. In various embodiments, exons 3 to 5 of the nucleotide sequence encoding for a functional factor IX protein has 80% identity to the sequence of SEQ ID NO. 6. In some embodiments, the nucleotide sequence of exons 3 to 5 has at least 82% identity to the sequence of SEQ ID NO. 6. In other embodiments, the nucleotide sequence of exons 3 to 5 has at least 84% identity to the sequence of SEQ ID NO. 6. In further embodiments, the nucleotide sequence of exons 3 to 5 has at least 86% identity to the sequence of SEQ ID NO. 6. In particular embodiments, the nucleotide sequence of exons 3 to 5 has at least 88% identity to the sequence of SEQ ID NO. 6. In some embodiments, the nucleotide sequence of exons 3 to 5 has at least 90% identity to the sequence of SEQ ID NO. 6. In other embodiments, the nucleotide sequence of exons 3 to 5 has at least 91% identity to the sequence of SEQ ID NO. 6. In further embodiments, the nucleotide sequence of exons 3 to 5 has at least 92% identity to the sequence of SEQ ID NO. 6. In particular embodiments, the nucleotide sequence of exons 3 to 5 has at least 93% identity to the sequence of SEQ ID NO. 6. In some embodiments, the nucleotide sequence of exons 3 to 5 has at least 94% identity to the sequence of SEQ ID NO. 6. In other embodiments, the nucleotide sequence of exons 3 to 5 has at least 95% identity to the sequence of SEQ ID NO. 6. In further embodiments, the nucleotide sequence of exons 3 to 5 has at least 96% identity to the sequence of SEQ ID NO. 6. In particular embodiments, the nucleotide sequence of exons 3 to 5 has at least 97% identity to the sequence of SEQ ID NO. 6. In some embodiments, the nucleotide sequence of exons 3 to 5 has at least 98% identity to the sequence of SEQ ID NO. 6. In other embodiments, the nucleotide sequence of exons 3 to 5 has at least 99% identity to the sequence of SEQ ID NO. 6. In preferred embodiments, the nucleotide sequence of exons 3 to 5 has the sequence of SEQ ID NO. 6. - The nucleotide sequence encoding for a functional FIX protein is preferably between 1335 and 1435 nucleotides in length. In some embodiments, the nucleotide sequence encoding for a functional FIX protein is between 1360 and 1410 nucleotides in length. In other embodiments, the nucleotide sequence encoding for a functional FIX protein is between 1375 and 1395 nucleotides in length. In particular embodiments, the nucleotide sequence encoding for a functional FIX protein is between 1380 and 1390 nucleotides in length.
- In some embodiments, the nucleotide sequence encoding for a functional FIX protein, including the intron sequence between
exons 1 and 2, has 80% identity to the sequence of SEQ ID NO. 3. In some embodiments, the nucleotide sequence has at least 82% identity to the sequence of SEQ ID NO. 3. In other embodiments, the nucleotide sequence has at least 84% identity to the sequence of SEQ ID NO. 3. In further embodiments, the nucleotide sequence has at least 86% identity to the sequence of SEQ ID NO. 3. In particular embodiments, the nucleotide sequence has at least 88% identity to the sequence of SEQ ID NO. 3. In some embodiments, the nucleotide sequence has at least 90% identity to the sequence of SEQ ID NO. 3. In other embodiments, the nucleotide sequence has at least 91% identity to the sequence of SEQ ID NO. 3. In further embodiments, the nucleotide sequence has at least 92% identity to the sequence of SEQ ID NO. 3. In particular embodiments, the nucleotide sequence has at least 93% identity to the sequence of SEQ ID NO. 3. In some embodiments, the nucleotide sequence has at least 94% identity to the sequence of SEQ ID NO. 3. In other embodiments, the nucleotide sequence has at least 95% identity to the sequence of SEQ ID NO. 3. In further embodiments, the nucleotide sequence has at least 96% identity to the sequence of SEQ ID NO. 3. In particular embodiments, the nucleotide sequence has at least 97% identity to the sequence of SEQ ID NO. 3. In some embodiments, the nucleotide sequence has at least 98% identity to the sequence of SEQ ID NO. 3. In other embodiments, the nucleotide sequence has at least 99% identity to the sequence of SEQ ID NO. 3. In preferred embodiments, the nucleotide sequence encoding for a functional FIX protein, including the intron sequence betweenexons 1 and 2, has the sequence of SEQ ID NO. 3. - Therefore, in preferred embodiments, the present invention provides a vector for expressing factor IX protein, the vector comprising a promoter, and a nucleotide sequence encoding for a functional factor IX protein, wherein an intron sequence is positioned between exon 1 and
exon 2 of the nucleotide sequence encoding for a functional factor IX protein, and wherein the factor IX nucleotide sequence, including the intron sequence, has at least 80% identity to the sequence of SEQ ID NO. 3. As described above, the percentage identity may be higher. - The promoter causes expression of the nucleotide sequence encoding for a functional factor IX protein. Any appropriate promoter may be used, such as HLP, LP1, HCR-hAAT, ApoE-hAAT, and LSP. These promoters are described in more detail in the following references: HLP: McIntosh J. et al., Blood 2013 Apr. 25, 121(17):3335-44; LP1: Nathwani et al., Blood. 2006 Apr. 1, 107(7): 2653-2661; HCR-hAAT: Miao et al., Mol Ther. 2000; 1: 522-532; ApoE-hAAT: Okuyama et al., Human Gene Therapy, 7, 637-645 (1996); and LSP: Wang et al., Proc Natl Acad Sci USA. 1999 Mar. 30, 96(7): 3906-3910. A preferred promoter is also described in WO 2011/005968. Preferably, the promoter is a liver specific promoter.
- The promoter preferably has a nucleotide sequence which has at least 80% identity to the sequence of SEQ ID NO. 4. In some embodiments, the nucleotide sequence has at least 82% identity to the sequence of SEQ ID NO. 4. In other embodiments, the nucleotide sequence has at least 84% identity to the sequence of SEQ ID NO. 4. In further embodiments, the nucleotide sequence has at least 86% identity to the sequence of SEQ ID NO. 4. In particular embodiments, the nucleotide sequence has at least 88% identity to the sequence of SEQ ID NO. 4. In some embodiments, the nucleotide sequence has at least 90% identity to the sequence of SEQ ID NO. 4. In other embodiments, the nucleotide sequence has at least 91% identity to the sequence of SEQ ID NO. 4. In further embodiments, the nucleotide sequence has at least 92% identity to the sequence of SEQ ID NO. 4. In particular embodiments, the nucleotide sequence has at least 93% identity to the sequence of SEQ ID NO. 4. In some embodiments, the nucleotide sequence has at least 94% identity to the sequence of SEQ ID NO. 4. In other embodiments, the nucleotide sequence has at least 95% identity to the sequence of SEQ ID NO. 4. In further embodiments, the nucleotide sequence has at least 96% identity to the sequence of SEQ ID NO. 4. In particular embodiments, the nucleotide sequence has at least 97% identity to the sequence of SEQ ID NO. 4. In some embodiments, the nucleotide sequence has at least 98% identity to the sequence of SEQ ID NO. 4. In other embodiments, the nucleotide sequence has at least 99% identity to the sequence of SEQ ID NO. 4. In preferred embodiments, the nucleotide sequence of the promoter is the sequence of SEQ ID NO. 4.
- The promoter having SEQ ID NO. 4 is a liver specific promoter which has been found to give particularly good expression in the liver. Whilst giving good expression, this promoter is also relatively small which allows more efficient packaging of the vector.
- The nucleotide sequence of the promoter is preferably between 300 and 400 nucleotides in length. In some embodiments, the nucleotide sequence of the promoter is between 330 and 380 nucleotides in length. In other embodiments, the nucleotide sequence of the promoter is between 345 and 365 nucleotides in length. In particular embodiments, the nucleotide sequence of the promoter is between 350 and 360 nucleotides in length.
- The vector may be any appropriate vector for expressing the FIX protein, including viral and non-viral vectors. Viral vectors include a parvovirus, an adenovirus, a retrovirus, a lentivirus or a herpes simplex virus. The parvovirus may be an adenovirus-associated virus (AAV). The vector is preferably a recombinant adeno-associated viral (rAAV) vector or a lentiviral vector. More preferably, the vector is an rAAV vector.
- A vector according to the invention may be a gene delivery vector. Such a gene delivery vector may be a viral gene delivery vector or a non-viral gene delivery vector.
- Accordingly, the present invention provides gene delivery vectors based on animal parvoviruses, in particular dependoviruses such as infectious human or simian AAV, and the components thereof (e.g., an animal parvovirus genome) for use as vectors for introduction and/or expression of a factor IX protein in a mammalian cell. The term “parvoviral” as used herein thus encompasses dependoviruses such as any type of AAV.
- Viruses of the Parvoviridae family are small DNA animal viruses. The family Parvoviridae may be divided between two subfamilies: the Parvovirinae, which infect vertebrates, and the Densovirinae, which infect insects. Members of the subfamily Parvovirinae are herein referred to as the parvoviruses and include the genus Dependovirus. As may be deduced from the name of their genus, members of the Dependovirus are unique in that they usually require coinfection with a helper virus such as adenovirus or herpes virus for productive infection in cell culture. The genus Dependovirus includes AAV, which normally infects humans (e.g., serotypes 1, 2, 3A, 3B, 4, 5, and 6) or primates (e.g., serotypes 1 and 4), and related viruses that infect other warm-blooded animals (e.g., bovine, canine, equine, and ovine adeno-associated viruses). Further information on parvoviruses and other members of the Parvoviridae is described in Kenneth I. Berns, “Parvoviridae: The Viruses and Their Replication,” Chapter 69 in Fields Virology (3d Ed. 1996). For convenience the present invention is further exemplified and described herein by reference to AAV. It is, however, understood that the invention is not limited to AAV but may equally be applied to other parvoviruses.
- The genomic organization of all known AAV serotypes is very similar. The genome of AAV is a linear, single-stranded DNA molecule that is less than about 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences for the non-structural replication (Rep) proteins and the structural (VP) proteins. The VP proteins (VP1, -2 and -3) form the capsid. The terminal 145 nt are self-complementary and are organized so that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. These hairpin structures function as an origin for viral DNA replication, serving as primers for the cellular DNA polymerase complex. Following wild type (wt) AAV infection in mammalian cells the Rep genes (i.e. encoding Rep78 and Rep52 proteins) are expressed from the 135 promoter and the P19 promoter, respectively, and both Rep proteins have a function in the replication of the viral genome. A splicing event in the Rep ORF results in the expression of actually four Rep proteins (i.e. Rep78, Rep68, Rep52 and Rep40). However, it has been shown that the unspliced mRNA, encoding Rep78 and Rep52 proteins, in mammalian cells are sufficient for AAV vector production. Also in insect cells the Rep78 and Rep52 proteins suffice for AAV vector production.
- In an AAV suitable for use as a gene therapy vector, the vector genome typically comprises a nucleic acid to be packaged for delivery to a target cell. According to this particular embodiment, the heterologous nucleotide sequence is located between the viral ITRs at either end of the vector genome. In further preferred embodiments, the parvovirus (e.g. AAV) cap genes and parvovirus (e.g. AAV) rep genes are deleted from the template genome (and thus from the virion DNA produced therefrom). This configuration maximizes the size of the nucleic acid sequence(s) that can be carried by the parvovirus capsid.
- According to this particular embodiment, the nucleic acid is located between the viral ITRs at either end of the substrate. It is possible for a parvoviral genome to function with only one ITR. Thus, in a gene therapy vector of the invention based on a parvovirus, the vector genome is flanked by at least one ITR, but, more typically, by two AAV ITRs (generally with one either side of the vector genome, i.e. one at the 5′ end and one at the 3′ end). There may be intervening sequences between the nucleic acid in the vector genome and one or more of the ITRs.
- Preferably, the nucleotide sequence encoding a functional factor IX protein (for expression in the mammalian cell) will be incorporated into a parvoviral genome located between two regular ITRs or located on either side of an ITR engineered with two D regions.
- AAV sequences that may be used in the present invention for the production of AAV gene therapy vectors can be derived from the genome of any AAV serotype. Generally, the AAV serotypes have genomic sequences of significant homology at the amino acid and the nucleic acid levels, provide an identical set of genetic functions, produce virions which are essentially physically and functionally equivalent, and replicate and assemble by practically identical mechanisms. For the genomic sequence of the various AAV serotypes and an overview of the genomic similarities see e.g. GenBank Accession number U89790; GenBank Accession number J01901; GenBank Accession number AF043303; GenBank Accession number AF085716; Chiorini et al, 1997; Srivastava et al, 1983; Chiorini et al, 1999; Rutledge et al, 1998; and Wu et al, 2000.
AAV serotype 1, 2, 3, 4, 5, 6, 7, 8 or 9 may be used in the present invention. However, AAV serotypes 1, 5 or 8 are preferred sources of AAV sequences for use in the context of the present invention. The sequences from the AAV serotypes may be mutated or engineered when being used in the production of gene therapy vectors. - Preferably, the AAV ITR sequences for use in the context of the present invention are derived from AAV1, AAV2, AAV4 and/or AAV6. Likewise, the Rep (Rep78 and Rep52) coding sequences are preferably derived from AAV1, AAV2, AAV4 and/or AAV6. The sequences coding for the VP1, VP2, and VP3 capsid proteins for use in the context of the present invention may however be taken from any of the known 42 serotypes, more preferably from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 or newly developed AAV-like particles obtained by e.g. capsid shuffling techniques and AAV capsid libraries.
- AAV Rep and ITR sequences are particularly conserved among most serotypes. The Rep78 proteins of various AAV serotypes are e.g. more than 89% identical and the total nucleotide sequence identity at the genome level between AAV2, AAV3A, AAV3B, and AAV6 is around 82% (Bantel-Schaal et al, 1999). Moreover, the Rep sequences and ITRs of many AAV serotypes are known to efficiently cross-complement (i.e., functionally substitute) corresponding sequences from other serotypes in production of AAV particles in mammalian cells. US 2003148506 reports that AAV Rep and ITR sequences also efficiently cross-complement other AAV Rep and ITR sequences in insect cells.
- The AAV VP proteins are known to determine the cellular tropicity of the AAV virion. The VP protein-encoding sequences are significantly less conserved than Rep proteins and genes among different AAV serotypes. The ability of Rep and ITR sequences to cross-complement corresponding sequences of other serotypes allows for the production of pseudotyped AAV particles comprising the capsid proteins of a serotype (e.g., AAV1, 5 or 8) and the Rep and/or ITR sequences of another AAV serotype (e.g., AAV2). Such pseudotyped rAAV particles are a part of the present invention.
- Modified “AAV” sequences also can be used in the context of the present invention, e.g. for the production of AAV gene therapy vectors. Such modified sequences e.g. include sequences having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more nucleotide and/or amino acid sequence identity (e.g., a sequence having about 75-99% nucleotide sequence identity) to an AAV1, AAV2, AAV3, AAV4, AAVS, AAV6, AAV7, AAV8 or AAVS ITR, Rep, or VP can be used in place of wild-type AAV ITR, Rep, or VP sequences.
- Although similar to other AAV serotypes in many respects, AAVS differs from other human and simian AAV serotypes more than other known human and simian serotypes. In view thereof, the production of rAAV5 can differ from production of other serotypes in insect cells. Where methods of the invention are employed to produce rAAV5, it is preferred that one or more constructs comprising, collectively in the case of more than one construct, a nucleotide sequence comprising an AAVS ITR, a nucleotide sequence comprises an AAVS Rep coding sequence (i.e. a nucleotide sequence comprises an AAVS Rep78). Such ITR and Rep sequences can be modified as desired to obtain efficient production of AAVS or pseudotyped AAVS vectors. For example, the start codon of the Rep sequences can be modified, VP splice sites can be modified or eliminated, and/or the VP1 start codon and nearby nucleotides can be modified to improve the production of AAV5 vectors.
- Thus, the viral capsid used in the invention may be from any parvovirus, either an autonomous parvovirus or dependovirus, as described above. Preferably, the viral capsid is an AAV capsid (e. g., AAV1, AAV2, AAV3, AAV4, AAV5 or AAV6 capsid). In general, the AAV1 capsid or AAV6 capsid are preferred. The choice of parvovirus capsid may be based on a number of considerations as known in the art, e.g., the target cell type, the desired level of expression, the nature of the heterologous nucleotide sequence to be expressed, issues related to viral production, and the like. For example, the AAV1 and AAV6 capsid may be advantageously employed for skeletal muscle; AAV1, AAV5 and AAV8 for the liver and cells of the central nervous system (e.g., brain); AAV5 for cells in the airway and lung or brain; AAV3 for bone marrow cells; and AAV4 for particular cells in the brain (e. g., appendable cells).
- It is within the technical skills of the skilled person to select the most appropriate virus, virus subtype or virus serotype. Some subtypes or serotypes may be more appropriate than others for a certain type of tissue.
- For example, liver-specific expression of a nucleic acid of the invention may advantageously be induced by AAV-mediated transduction of liver cells. Liver is amenable to AAV-mediated transduction, and different serotypes may be used (for example, AAV1, AAV5 or AAV8). Transduction of muscle may be accomplished by administration of an AAV encoding a nucleic acid via the blood stream. Thus, intravenous or intra-arterial administration is applicable.
- A parvovirus gene therapy vector prepared according to the invention may be a “hybrid” particle in which the viral ITRs and viral capsid are from different parvoviruses. Preferably, the viral TRs and capsid are from different serotypes of AAV. Likewise, the parvovirus may have a “chimeric” capsid (e. g., containing sequences from different parvoviruses, preferably different AAV serotypes) or a “targeted” capsid (e. g., a directed tropism).
- In the context of the invention “at least one parvoviral ITR nucleotide sequence” is understood to mean a palindromic sequence, comprising mostly complementary, symmetrically arranged sequences also referred to as “A,” “B,” and “C” regions. The ITR functions as an origin of replication, a site having a “cis” role in replication, i.e., being a recognition site for trans-acting replication proteins such as e.g. Rep 78 (or Rep68) which recognize the palindrome and specific sequences internal to the palindrome. One exception to the symmetry of the ITR sequence is the “D” region of the ITR. It is unique (not having a complement within one ITR). Nicking of single-stranded DNA occurs at the junction between the A and D regions. It is the region where new DNA synthesis initiates. The D region normally sits to one side of the palindrome and provides directionality to the nucleic acid replication step. A parvovirus replicating in a mammalian cell typically has two ITR sequences. It is, however, possible to engineer an ITR so that binding sites are on both strands of the A regions and D regions are located symmetrically, one on each side of the palindrome. On a double-stranded circular DNA template (e.g., a plasmid), the Rep78- or Rep68-assisted nucleic acid replication then proceeds in both directions and a single ITR suffices for parvoviral replication of a circular vector. Thus, one ITR nucleotide sequence can be used in the context of the present invention. Preferably, however, two or another even number of regular ITRs are used. Most preferably, two ITR sequences are used. A preferred parvoviral ITR is an AAV ITR. For safety reasons it may be desirable to construct a parvoviral (AAV) vector that is unable to further propagate after initial introduction into a cell. Such a safety mechanism for limiting undesirable vector propagation in a recipient may be provided by using AAV with a chimeric ITR as described in US 2003148506.
- Those skilled in the art will appreciate that the viral Rep protein(s) used for producing an AAV vector of the invention may be selected with consideration for the source of the viral ITRs. For example, the AAVS ITR typically interacts more efficiently with the AAVS Rep protein, although it is not necessary that the serotype of ITR and Rep protein(s) are matched.
- The ITR(s) used in the invention are typically functional, i.e. they may be fully resolvable and are preferably AAV sequences, with
serotypes 1, 2, 3, 4, 5 or 6 being preferred. Resolvable AAV ITRs according to the present invention need not have a wild-type ITR sequence (e. g., a wild-type sequence may be altered by insertion, deletion, truncation or missense mutations), as long as the ITR mediates the desired functions, e. g., virus packaging, integration, and/or provirus rescue, and the like. - Advantageously, by using a gene therapy vector as compared with previous approaches, the restoration of protein synthesis, i.e. factor IX synthesis, is a characteristic that the transduced cells acquire permanently or for a sustained period of time, thus avoiding the need for continuous administration to achieve a therapeutic effect.
- Accordingly, the vectors of the invention therefore represent a tool for the development of strategies for the in vivo delivery of a FIX nucleotide sequence, by engineering the nucleic acid within a gene therapy vector that efficiently transduces an appropriate cell type, such as a liver cell.
- Preferably, the vector is a single stranded vector rather than a self-complementary vector. Surprisingly, this has been shown to give better protein expression.
- The vector may further comprise a poly A tail. Preferably, this is positioned downstream of the nucleotide sequence encoding for a functional FIX protein. Preferably, the poly A tail is a bovine growth hormone poly A tail (bGHpA). Preferably, this is between 250 and 270 nucleotides in length.
- In a preferred embodiment, the vector comprises a nucleotide sequence which has 80% identity to the sequence of SEQ ID NO. 5. In some embodiments, the nucleotide sequence has at least 82% identity to the sequence of SEQ ID NO. 5. In other embodiments, the nucleotide sequence has at least 84% identity to the sequence of SEQ ID NO. 5. In further embodiments, the nucleotide sequence has at least 86% identity to the sequence of SEQ ID NO. 5. In particular embodiments, the nucleotide sequence has at least 88% identity to the sequence of SEQ ID NO. 5. In some embodiments, the nucleotide sequence has at least 90% identity to the sequence of SEQ ID NO. 5. In other embodiments, the nucleotide sequence has at least 91% identity to the sequence of SEQ ID NO. 5. In further embodiments, the nucleotide sequence has at least 92% identity to the sequence of SEQ ID NO. 5. In particular embodiments, the nucleotide sequence has at least 93% identity to the sequence of SEQ ID NO. 5. In some embodiments, the nucleotide sequence has at least 94% identity to the sequence of SEQ ID NO. 5. In other embodiments, the nucleotide sequence has at least 95% identity to the sequence of SEQ ID NO. 5. In further embodiments, the nucleotide sequence has at least 96% identity to the sequence of SEQ ID NO. 5. In particular embodiments, the nucleotide sequence has at least 97% identity to the sequence of SEQ ID NO. 5. In some embodiments, the nucleotide sequence has at least 98% identity to the sequence of SEQ ID NO. 5. In other embodiments, the nucleotide sequence has at least 99% identity to the sequence of SEQ ID NO. 5. In preferred embodiments, the vector comprises the nucleotide sequence of SEQ ID NO. 5.
- In another aspect of the invention, there is provided a nucleic acid molecule comprising a nucleotide sequence encoding for a functional factor IX protein, wherein exons 3 to 5 of the nucleotide sequence have at least 80% identity to the sequence of SEQ ID NO. 6.
- The invention also provides a vector comprising a nucleotide sequence encoding for a functional factor IX protein, wherein exons 3 to 5 of the nucleotide sequence have at least 80% identity to the sequence of SEQ ID NO. 6. The vector will comprise other elements to allow the functional FIX protein to be expressed such as a promoter. Such elements are well known to a person skilled in the art.
- Additional features relating to the nucleotide sequence encoding for a functional factor IX protein, and exons 3 to 5 of the sequence, are described above.
- In another aspect of the invention, there is provided a nucleic acid molecule comprising a nucleotide sequence encoding for a functional factor IX protein, the nucleotide sequence having at least 80% identity to the sequence of SEQ ID NO. 2.
- The invention also provides a vector comprising a nucleotide sequence encoding for a functional factor IX protein, the nucleotide sequence having at least 80% identity to the sequence of SEQ ID NO. 2. The vector will comprise other elements to allow the functional FIX protein to be expressed such as a promoter. Such elements are well known to a person skilled in the art.
- Additional features relating to the nucleotide sequence encoding for a functional factor IX protein are described above.
- In another aspect of the invention, there is provided a nucleic acid molecule comprising a nucleotide sequence encoding for a functional factor IX protein and containing an intron sequence positioned between exon 1 and
exon 2 of the factor IX sequence, wherein the nucleotide sequence has at least 80% identity to the sequence of SEQ ID NO. 3. - Additional features relating to the nucleotide sequence encoding for a functional factor IX protein and which contains an intron sequence are described above.
- In another aspect of the invention, there is provided a nucleic acid molecule comprising an intron sequence having at least 80% identity to the sequence of SEQ ID NO. 1.
- Additional features relating to the intron sequence are described above.
- In another aspect of the invention, there is provided a nucleic acid molecule comprising a promoter, wherein the promoter has a nucleotide sequence having at least 80% identity to the sequence of SEQ ID NO. 4.
- Additional features relating to the promoter sequence are described above.
- Preferably, the nucleic acids described above are isolated.
- It would be well with the capabilities of a skilled person to produce the nucleic acid molecules described above. This could be done, for example, using chemical synthesis of a given sequence.
- Further, a skilled person would readily be able to determine whether a nucleic acid expresses a functional protein. Suitable methods would be apparent to those skilled in the art. For example, one suitable in vitro method involves inserting the nucleic acid into a vector, such as a lentiviral or an AAV vector, transducing host cells, such as 293T or HeLa cells, with the vector, and assaying for factor IX activity. Alternatively, a suitable in vivo method involves transducing a vector containing the nucleic acid into haemophiliac mice and assaying for functional factor IX in the plasma of the mice. Suitable methods are described in more detail below.
- The nucleic acid can be any type of nucleic acid composed of nucleotides. The nucleic acid should be able to be expressed so that a protein is produced. Preferably, the nucleic acid is DNA or RNA.
- The invention also provides a host cell comprising any one of the nucleic acid molecules or vectors described above. Preferably, the vector is capable of expressing the FIX nucleotide sequence in the host. The host may be any suitable host.
- As used herein, the term “host” refers to organisms and/or cells which harbour a nucleic acid molecule or a vector of the invention, as well as organisms and/or cells that are suitable for use in expressing a recombinant gene or protein. It is not intended that the present invention be limited to any particular type of cell or organism. Indeed, it is contemplated that any suitable organism and/or cell will find use in the present invention as a host. A host cell may be in the form of a single cell, a population of similar or different cells, for example in the form of a culture (such as a liquid culture or a culture on a solid substrate), an organism or part thereof
- A host cell according to the invention may permit the expression of a nucleic acid molecule of the invention. Thus, the host cell may be, for example, a bacterial, a yeast, an insect or a mammalian cell.
- In addition, the invention provides a transgenic animal comprising cells comprising the nucleic acid molecule encoding for a functional FIX protein described above or a vector described above. Preferably the animal is a non-human mammal, especially a primate. Alternatively, the animal may be a rodent, especially a mouse; or may be canine, feline, ovine or porcine.
- In one aspect, the invention provides a pharmaceutical composition comprising a nucleic acid molecule or a vector of the invention and one or more pharmaceutically acceptable excipients. The one or more excipients include carriers, diluents and/or other medicinal agents, pharmaceutical agents or adjuvants, etc.
- The invention also provides a method of treating haemophilia B comprising administering a therapeutically effective amount of a vector as described above to a patient suffering from haemophilia B. Preferably, the patient is human.
- When haemophilia B is “treated” in the above method, this means that one or more symptoms of haemophilia are ameliorated. It does not mean that the symptoms of haemophilia are completely remedied so that they are no longer present in the patient, although in some methods, this may be the case. The method of treating results in one or more of the symptoms of haemophilia B being less severe than before treatment.
- A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as raising the level of functional factor IX in a subject (so as to lead to functional factor IX production to a level sufficient to ameliorate the symptoms of haemophilia B).
- Delivery of a nucleic acid or vector of the invention to a host cell in vivo may result in an increase of functional factor IX in the host, for example to a level that ameliorates one or more symptoms of haemophilia B.
- The level of naturally occurring factor IX in a subject suffering from haemophilia B varies depending on the severity of the haemophilia. Patients with a severe form of the disease have factor IX levels of less than about 1% of the level found in a normal healthy subject (referred to herein as “a normal level”). It has been found that when the method of treatment of the invention is used, it can cause an increase in the level of functional factor IX to at least about 1% of normal levels. In a subject suffering from haemophilia B, an increase in circulating FIX to >1% of normal levels can significantly ameliorate the severe bleeding phenotype. In some embodiments, the method of treatment of the invention causes an increase in the level of functional factor IX to at least about 2%, at least about 3%, at least about 4%, at least about 10%, at least about 15%, at least about 20% or at least about 25% of normal levels. In a particular embodiment, the method of treatment of the invention causes an increase in the level of functional factor IX to at least about 30% of normal levels. This level of increase would virtually normalise coagulation of blood in subjects suffering haemophilia B. Such subjects are unlikely to require factor IX concentrates following trauma or during surgery.
- In one embodiment, the method of treatment of the invention causes an increase in the level of functional factor IX to, at most, normal levels.
- The level of functional factor IX can be measured relatively easily and methods for measuring factor IX levels are well known to those skilled in the art. Many clotting assays are available, including chromogenic and clotting based assays. ELISA tests are also widely available.
- Further, the invention provides the nucleic acid molecule encoding for a functional FIX protein as described above or a vector as described above for use in therapy, for example, in the treatment of haemophilia B.
- In addition, the invention provides the use of the nucleic acid molecule encoding for a functional FIX protein as described above or a vector as described above in the manufacture of a medicament for treating haemophilia B.
- The invention also provided a method for delivery of a nucleotide sequence encoding a functional FIX protein to a subject, which method comprises administering to the said subject a nucleic acid molecule encoding a functional FIX protein as described above or a vector as described above.
- In the description above, the term “identity” is used to refer to the similarity of two sequences. For the purpose of this invention, it is defined here that in order to determine the percent identity of two nucleotide sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first nucleic acid for optimal alignment with a second amino or nucleic acid sequence). The nucleotide residues at nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical positions/total number of positions (i.e. overlapping positions)×100). Preferably, the two sequences are the same length. A sequence comparison is typically carried out over the entire length of the two sequences being compared.
- The skilled person will be aware of the fact that several different computer programs are available to determine the homology or identity between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two nucleic acid sequences is determined using the sequence alignment software Clone Manager 9 (Sci-Ed software—www.scied.com) using global DNA alignment; parameters: both strands; scoring matrix: linear (
mismatch 2, OpenGap 4, ExtGap 1). - Alternatively, the percent identity between two amino acid or nucleic acid sequences is determined using the Needleman and Wunsch (1970) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at http://www.accelrys.com/products/gcg/), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
- The nucleic acid sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTN programs (version 2.0) of Altschul, et al, 1990. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g. BLASTN) can be used. See the homepage of the National Center for Biotechnology Information at hill): //www.ncbi.nlm.nih.gov/.
- All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.
- A skilled person will appreciate that all aspects of the invention, whether they relate to, for example, the nucleic acid, the vector, the host cell or the use, are equally applicable to all other aspects of the invention. In particular, aspects of the method of treatment, for example, the administration of the nucleic acid or vector, may have been described in greater detail than in some of the other aspects of the invention, for example, relating to the use of the nucleic acid or vector for treating haemophilia B. However, the skilled person will appreciate where more detailed information has been given for a particular aspect of the invention, this information is generally equally applicable to other aspects of the invention. Further, the skilled person will also appreciate that the description relating to the method of treatment is equally applicable to the use of the nucleic acid or vector in treating haemophilia B.
- The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:
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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. -
FIG. 2 shows FIX expression of the two constructs shown inFIG. 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. -
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. -
FIG. 4 shows FIX expression of the two constructs shown inFIG. 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. -
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. -
FIG. 6 shows FIX expression of the two constructs shown inFIG. 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. - 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 16% 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.
- 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 lel 1 vg was administered into the tail vein of 4-6 week old male C57B1/6 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 ). - 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 C57B1/6 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 inFIG. 4 . - 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 titered 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 C57B1/6 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. - SEQ ID NO. 1—Nucleotide sequence of truncated intron (TI).
- SEQ ID NO. 2—Nucleotide sequence of codon optimised FIX. Features: FIX Exon 1: 1-88; FIX Exons 2-5: 89-1386.
- 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.
- SEQ ID NO. 4—Nucleotide sequence of promoter HLP2.
- 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.
- SEQ ID NO. 6—Nucleotide sequence of codon optimised exons 3 to 5 of FIX.
Claims (14)
1.-46. (canceled)
47. 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 the functional factor IX protein, and wherein:
the intron sequence has at least 80% identity to the sequence of SEQ ID NO. 1,
the intron sequence has at least 95% identity to the sequence of SEQ ID NO. 1, or
the intron sequence has the sequence of SEQ ID NO. 1.
48. The vector of claim 47 , wherein:
the promoter has a nucleotide sequence which has at least 80% identity to the sequence of SEQ ID NO. 4,
the promoter has a nucleotide sequence which has at least 95% identity to the sequence of SEQ ID NO. 4, or
the promoter has the nucleotide sequence of SEQ ID NO. 4.
49. The vector of claim 47 , wherein:
the nucleotide sequence encoding for the functional protein has at least 80% identity to the sequence of SEQ ID NO. 2,
the nucleotide sequence encoding for the functional FIX protein has at least 95% identity to the sequence of SEQ ID NO. 2, or
the nucleotide sequence encoding for the functional FIX protein has the sequence of SEQ ID NO. 2.
50. The vector of claim 47 , wherein:
the nucleotide sequence encoding the functional FIX protein has at least 80% identity to the sequence of SEQ ID NO. 6,
the nucleotide sequence encoding the functional FIX protein has at least 95% identity to the sequence of SEQ ID NO. 6, or
the nucleotide sequence encoding the functional FIX protein has the sequence of SEQ ID NO. 6.
51. The vector of claim 47 , wherein:
the nucleotide sequence encoding for the functional FIX protein, including the intron sequence between exon 1 and 2, has 80% identity to the sequence of SEQ ID NO. 3,
the nucleotide sequence encoding for the functional FIX protein, including the intron sequence between exon 1 and 2, has 95% identity to the sequence of SEQ ID NO. 3, or
the nucleotide sequence encoding for the functional FIX protein, including the intron sequence between exon 1 and 2, has the sequence of SEQ ID NO. 3.
52. The vector of claim 47 , wherein the nucleotide sequence encoding for the functional factor IX protein comprises:
a nucleotide sequence that has 80% identity, 95% identity, or 100% identity to nucleotides 1-88 (exon 1) of Genbank accession number J00137.1; or
a nucleotide sequence that has 80% identity, 95% identity; or 100% identity to nucleotides 89-197 (exon 2, partial) of Genbank accession number J00137.1.
53. The vector of claim 52 , wherein the nucleotide sequence encoding for the functional factor IX protein is codon optimized.
54. The vector of claim 53 , wherein the nucleotide sequence encoding for the functional factor IX protein comprises a nucleotide sequence that has 80% identity to Genbank accession number J00137.1
55. The vector of claim 47 ; wherein:
the vector comprises a nucleotide sequence which has 80% identity to the sequence of SEQ ID NO. 5,
the vector comprises a nucleotide sequence which has 95% identity to the sequence of SEQ ID NO. 5, or
the vector comprises a nucleotide sequence which has the sequence of SEQ ID NO. 5.
56. The vector of claim 47 , wherein:
the intron sequence has at least 95% identity to the sequence of SEQ ID NO. 1,
the nucleotide sequence encoding for the functional FIX protein has at least 95% identity to the sequence of SEQ ID NO. 2, and
the promoter has a nucleotide sequence which has at least 95% identity to the sequence of SEQ ID NO. 4, and
wherein the vector is a single stranded vector.
57. The vector of claim 47 , wherein the vector:
is an AAV vector,
is a single stranded vector, or
further comprises a bovine growth hormone poly A tail.
58. A pharmaceutical composition comprising the vector of claim 47 , and one or more pharmaceutically acceptable excipients.
59. A method of treating haemophilia B comprising administering a therapeutically effective amount of the vector of claim 47 to a patient suffering from haemophilia B.
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB201420139D0 (en) | 2014-11-12 | 2014-12-24 | Ucl Business Plc | Factor IX gene therapy |
CA2990193A1 (en) | 2015-06-23 | 2016-12-29 | The Children's Hospital Of Philadelphia | Modified factor ix, and compositions, methods and uses for gene transfer to cells, organs and tissues |
GB201608046D0 (en) | 2016-05-09 | 2016-06-22 | Cambridge Entpr Ltd And Syndey Children S Hospitals Network Randwick And Westmead Incorporating The | Treatment of complement-mediated disorders |
KR102625470B1 (en) * | 2017-05-31 | 2024-01-16 | 더 유니버시티 오브 노쓰 캐롤라이나 엣 채플 힐 | Optimized human coagulation factor IX gene expression cassette and use thereof |
US20210236644A1 (en) | 2017-11-10 | 2021-08-05 | Cocoon Biotech Inc. | Ocular applications of silk-based products |
WO2020039183A1 (en) | 2018-08-20 | 2020-02-27 | Ucl Business Plc | Factor ix encoding nucleotides |
GB2576508A (en) * | 2018-08-20 | 2020-02-26 | Ucl Business Plc | Factor IX encoding nucleotides |
US10842885B2 (en) | 2018-08-20 | 2020-11-24 | Ucl Business Ltd | Factor IX encoding nucleotides |
LT3722434T (en) | 2019-04-12 | 2022-11-10 | Freeline Therapeutics Limited | Plasmid system |
CN113891942A (en) | 2019-04-12 | 2022-01-04 | 自由行疗法有限公司 | Plasmid system |
KR20220093177A (en) * | 2019-11-01 | 2022-07-05 | 프리라인 테라퓨틱스 리미티드 | transcriptional regulatory elements |
WO2021142376A1 (en) | 2020-01-08 | 2021-07-15 | Obsidian Therapeutics, Inc. | Compositions and methods for tunable regulation of transcription |
GB202016254D0 (en) | 2020-10-13 | 2020-11-25 | Freeline Therapeutics Ltd | Plasmid system |
Family Cites Families (148)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5839443A (en) | 1996-05-16 | 1998-11-24 | The Trustees Of Columbia University In The City Of New York | Method for inhibiting thrombosis in a patient whose blood is subjected to extracorporeal circulation |
US6315995B1 (en) | 1996-09-27 | 2001-11-13 | The Trustees Of Columbia University In The City Of New York | Methods for treating an ischemic disorder and improving stroke outcome |
WO1998041240A1 (en) | 1997-03-14 | 1998-09-24 | The Children's Hospital Of Philadelphia | Methods and compositions for use in gene therapy for treatment of hemophilia |
WO1999003496A1 (en) | 1997-07-21 | 1999-01-28 | The University Of North Carolina At Chapel Hill | Factor ix antihemophilic factor with increased clotting activity |
WO1999049803A1 (en) | 1998-04-01 | 1999-10-07 | The Trustees Of Columbia University In The City Of New York | Method for inhibiting thrombosis in a patient whose blood is subjected to extracorporeal circulation |
US20020065236A1 (en) | 1998-09-09 | 2002-05-30 | Yew Nelson S. | CpG reduced plasmids and viral vectors |
EP1832657B1 (en) | 1998-09-09 | 2012-10-24 | Genzyme Corporation | Methylation of plasmid vectors |
EP1010762A1 (en) | 1998-12-02 | 2000-06-21 | Aventis Behring Gesellschaft mit beschränkter Haftung | DNA constructs of blood clotting factors and P-Selectin |
EP1026250A1 (en) | 1998-12-02 | 2000-08-09 | Aventis Behring GmbH | DNA-constructs of blood clotting factors and P-selectin |
AU780854B2 (en) | 1999-02-19 | 2005-04-21 | Octagene Gmbh | Hormone-hormone receptor complexes and nucleic acid constructs and their use in gene therapy |
WO2000054787A1 (en) | 1999-03-16 | 2000-09-21 | The Children's Hospital Of Philadelphia | Enhanced gamma-carboxylation of recombinant vitamin k-dependent clotting factors |
EP1048736A1 (en) | 1999-04-27 | 2000-11-02 | Aventis Behring Gesellschaft mit beschränkter Haftung | DNA-construct for the tissue specific expression of a blood coagulation factor |
EP1038959A1 (en) | 1999-03-17 | 2000-09-27 | Aventis Behring Gesellschaft mit beschränkter Haftung | Factor VIII without B-domain, comprising one or more insertions of a truncated intron I of factor IX |
EP1048726B1 (en) | 1999-04-27 | 2006-07-26 | Négrier, Claude | Modified factor VIII cDNA |
WO2002064799A2 (en) | 1999-09-28 | 2002-08-22 | Transkaryotic Therapies, Inc. | Optimized messenger rna |
WO2001036620A2 (en) | 1999-11-16 | 2001-05-25 | Genzyme Corporation | Vectors and transgenies with regulatory elements for gene delivery to the liver |
DK1259265T3 (en) | 2000-03-03 | 2011-07-11 | Genetronics Inc | Nucleic acid formulations for delivery of genes |
AU2001249389A1 (en) | 2000-03-22 | 2001-10-03 | The Children's Hospital Of Philadelphia | Modified blood clotting factors and methods of use |
US7572619B2 (en) | 2000-03-22 | 2009-08-11 | Octagene Gmbh | Recombinant blood clotting factors |
US20020086427A1 (en) | 2000-03-23 | 2002-07-04 | Leiden Jeffrey M. | Inducible eukaryotic expression system that regulates protein translation |
EP1276849A4 (en) | 2000-04-12 | 2004-06-09 | Human Genome Sciences Inc | Albumin fusion proteins |
US7351813B2 (en) | 2000-06-20 | 2008-04-01 | The Board Of Trustees Of The Leland Stanford Junior University | Liver-specific gene expression cassettes, and methods of use |
CN1148228C (en) | 2000-08-30 | 2004-05-05 | 夏家辉 | Gene medicine for treating hemophilia B and its preparing process |
AU2002226028A1 (en) | 2000-11-14 | 2002-05-27 | Board Of Regents, Unversity Of Texas Systems | Mutant human factor ix with an increased resistance to inhibition by heparin |
EP1359936B1 (en) | 2001-02-05 | 2008-02-20 | Novo Nordisk Health Care AG | Combined use of factor vii polypeptides and factor viii polypeptides |
EP1379134A4 (en) | 2001-03-14 | 2006-04-05 | Genteric Inc | Recombinant adeno-assocaited virus-mediated gene transfer via retroductal infusion of virions |
JP2004532039A (en) | 2001-03-26 | 2004-10-21 | ザ ボード オブ トラスティーズ オブ ザ リーランド スタンフォード ジュニア ユニバーシティ | Helper-dependent adenovirus vector system and methods of using the system |
EP1478751A4 (en) | 2001-03-30 | 2005-10-19 | Avigenics Inc | Avian lysozyme promoter |
AU2002317771A1 (en) | 2001-06-05 | 2002-12-16 | Cellectis | Methods for modifying the cpg content of polynucleotides |
US20040254106A1 (en) | 2001-09-04 | 2004-12-16 | Carr Francis J. | Modified factor ix |
US7312374B2 (en) | 2001-09-18 | 2007-12-25 | Avigenics, Inc | Production of a transgenic avian by cytoplasmic injection |
US7550650B2 (en) | 2001-09-18 | 2009-06-23 | Synageva Biopharma Corp. | Production of a transgenic avian by cytoplasmic injection |
US7179617B2 (en) | 2001-10-10 | 2007-02-20 | Neose Technologies, Inc. | Factor IX: remolding and glycoconjugation of Factor IX |
CA2361462A1 (en) | 2001-11-07 | 2003-05-08 | Katherine A. High | Induction of tolerance to a therapeutic polypeptide |
US6723551B2 (en) | 2001-11-09 | 2004-04-20 | The United States Of America As Represented By The Department Of Health And Human Services | Production of adeno-associated virus in insect cells |
US20040210954A1 (en) | 2003-03-07 | 2004-10-21 | Alex Harvey | Integrase mediated avian transgenesis |
US6875588B2 (en) | 2001-11-30 | 2005-04-05 | Avigenics, Inc. | Ovomucoid promoter and methods of use |
WO2003066867A2 (en) | 2002-02-06 | 2003-08-14 | Artemis Pharmaceuticals Gmbh | Genetically engineered phic31-integrase genes |
NZ561656A (en) | 2002-05-01 | 2009-03-31 | Univ Florida | Improved rAAV expression systems for genetic modification of specific capsid proteins |
US20050034186A1 (en) | 2003-03-07 | 2005-02-10 | Harvey Alex J. | Site specific nucleic acid integration |
WO2004092351A2 (en) | 2003-03-27 | 2004-10-28 | Avigenics, Inc. | Production of a transgenic avian by cytoplasmic injection |
WO2006127896A2 (en) | 2005-05-25 | 2006-11-30 | Neose Technologies, Inc. | Glycopegylated factor ix |
EP1628628A4 (en) | 2003-05-05 | 2007-03-21 | Genzyme Corp | Methods of reducing an immune response |
WO2005040215A2 (en) | 2003-06-06 | 2005-05-06 | Avigenics, Inc. | Ovomucoid promoters and mehtods of use |
WO2005037226A2 (en) | 2003-10-17 | 2005-04-28 | Georgia Tech Research Corporation | Genetically engineered enteroendocrine cells for treating glucose-related metabolic disorders |
EP2311437A1 (en) | 2003-12-19 | 2011-04-20 | Novo Nordisk Health Care AG | Stabilised compositions of factor VII polypeptides |
JP3854283B2 (en) | 2004-04-26 | 2006-12-06 | 株式会社アイディエス | Straightening opening device for peelable cap |
WO2006015789A2 (en) | 2004-08-03 | 2006-02-16 | Geneart Ag | Method for modulating gene expression by modifying the cpg content |
ES2346072T3 (en) | 2004-08-17 | 2010-10-08 | Csl Behring Gmbh | DEPENDENT MODIFIED POLYPEPTIDES OF VITAMIN K. |
WO2006026238A2 (en) | 2004-08-25 | 2006-03-09 | Avigenics, Inc. | Rna interference in avians |
ATE549037T1 (en) | 2004-09-22 | 2012-03-15 | St Jude Childrens Res Hospital | IMPROVED EXPRESSION OF FACTOR-IX IN GENE THERAPY VECTORS |
WO2006093847A1 (en) | 2005-02-28 | 2006-09-08 | Avigenics, Inc. | Artificial chromosomes and transchromosomic avians |
EP1707634A1 (en) | 2005-03-29 | 2006-10-04 | Octapharma AG | Method for isolation of recombinantly produced proteins |
EP2359865B1 (en) | 2005-04-07 | 2013-10-02 | The Trustees of The University of Pennsylvania | Method of increasing the function of an AAV vector |
AU2006296840A1 (en) | 2005-09-30 | 2007-04-05 | Zgene A/S | Dekkera/Brettanomyces cytosine deaminases and their use |
US20090304641A1 (en) | 2005-10-17 | 2009-12-10 | Children's Hospital Boston | Methods and Compositions for Regulating Gene Expression |
WO2007046703A2 (en) | 2005-10-20 | 2007-04-26 | Amsterdam Molecular Therapeutics B.V. | Improved aav vectors produced in insect cells |
WO2007120533A2 (en) | 2006-03-30 | 2007-10-25 | The Board Of Trustees Of The Leland Stanford Junior University | Minigene expression cassette |
EP2007795B1 (en) | 2006-03-30 | 2016-11-16 | The Board Of Trustees Of The Leland Stanford Junior University | Aav capsid proteins |
EP2395099A3 (en) | 2006-05-02 | 2012-05-16 | Allozyne, Inc. | Amino acid substituted molecules |
US20080096819A1 (en) | 2006-05-02 | 2008-04-24 | Allozyne, Inc. | Amino acid substituted molecules |
EP2213733A3 (en) | 2006-05-24 | 2010-12-29 | Novo Nordisk Health Care AG | Factor IX analogues having prolonged in vivo half life |
EP2037892B1 (en) | 2006-06-19 | 2015-03-18 | Asklepios Biopharmaceutical, Inc. | Modified factor viii and factor ix genes and vectors for gene therapy |
EP2423307A1 (en) | 2006-06-19 | 2012-02-29 | Catalyst Biosciences, Inc. | Modified coagulation factor IV polypeptides and use thereof for treatment |
EP3023500B1 (en) | 2006-06-21 | 2020-02-12 | uniQure IP B.V. | Insect cells for the production of aav vectors |
US7700734B2 (en) | 2007-01-09 | 2010-04-20 | Shu-Wha Lin | Recombinant human factor IX and use thereof |
WO2008091311A1 (en) | 2007-01-26 | 2008-07-31 | Synageva Biopharma Corp | Transgene expression in avians |
WO2008092644A2 (en) | 2007-02-01 | 2008-08-07 | Baxter International Inc. | Fviii-independent fix-mutant proteins for hemophilia a treatment |
AU2008209985A1 (en) | 2007-02-01 | 2008-08-07 | Baxter Healthcare S.A. | Improved fix-mutant proteins for hemophilia B treatment |
US9725485B2 (en) | 2012-05-15 | 2017-08-08 | University Of Florida Research Foundation, Inc. | AAV vectors with high transduction efficiency and uses thereof for gene therapy |
EP3492596A1 (en) | 2007-04-09 | 2019-06-05 | University of Florida Research Foundation, Inc. | Raav vector compositions having tyrosine-modified capsid proteins and methods for use |
KR101229418B1 (en) * | 2007-06-15 | 2013-02-05 | 재단법인 목암생명공학연구소 | Method for manufacturing active recombinant blood coagulation factor ix |
PT2173888T (en) | 2007-07-26 | 2016-11-17 | Uniqure Ip Bv | Baculoviral vectors comprising repeated coding sequences with differential codon biases |
ES2426091T3 (en) | 2007-09-19 | 2013-10-21 | Uniqure Ip B.V. | Use of AAV replication machinery for improved protein production |
EP2209487A4 (en) | 2007-10-15 | 2012-06-20 | Univ North Carolina | Human factor ix variants with an extended half life |
EP2220218A4 (en) | 2007-11-02 | 2010-12-08 | Scripps Research Inst | A genetically encoded boronate amino acid |
US9249408B2 (en) | 2007-11-02 | 2016-02-02 | The Scripps Research Institute | Directed evolution using proteins comprising unnatural amino acids |
CN101952440B (en) | 2008-02-14 | 2012-11-14 | 财团法人牧岩生命工学研究所 | Expression vector suitable for expression of a coding sequence for gene therapy |
RU2010146387A (en) | 2008-04-16 | 2012-05-27 | БАЙЕР ХЕЛСКЕР ЛЛСи (US) | MODIFIED POLYPEPTIDES OF FACTOR IX AND THEIR APPLICATION |
CA2721362A1 (en) | 2008-04-16 | 2009-11-19 | Bayer Healthcare Llc | Site-directed modification of factor ix |
EP2268807A2 (en) | 2008-04-21 | 2011-01-05 | Novo Nordisk A/S | Hyperglycosylated human coagulation factor ix |
EP2149603A1 (en) | 2008-07-28 | 2010-02-03 | DRK-Blutspendedienst Baden-Württemberg-Hessen gGmbH | Factor IX variants with clotting activity in absence of their cofactor and their use for treating bleeding disorders |
US9249405B2 (en) | 2008-09-15 | 2016-02-02 | Paolo Simioni | Factor IX polypeptide mutant, its uses and a method for its production |
WO2010062322A2 (en) | 2008-10-27 | 2010-06-03 | Massachusetts Institute Of Technology | Modulation of the immune response |
DK2364362T3 (en) | 2008-11-12 | 2016-01-25 | Ospedale San Raffaele Srl | Gene vector FOR INDUCTION OF IMMUNE TOLERANCE transgene |
US20110070241A1 (en) | 2009-06-30 | 2011-03-24 | Duke University | Methods for modulating immune responses to aav gene therapy vectors |
GB0911870D0 (en) | 2009-07-08 | 2009-08-19 | Ucl Business Plc | Optimised coding sequence and promoter |
WO2011011841A1 (en) | 2009-07-31 | 2011-02-03 | Fundação Hemocentro de Ribeirão Preto | Human blood coagulation factor ix recombinant protein, composition, use of a factor ix recombinant protein, use of a composition, method of obtaining human blood coagulation factor ix recombinant protein and use of the factor ix recombinant protein |
EA201290069A1 (en) | 2009-07-31 | 2012-07-30 | Байер Хелскер Ллс | MODIFIED POLYPEPTIDES OF FACTOR IX AND THEIR APPLICATION |
EP2497830A1 (en) | 2009-11-05 | 2012-09-12 | Proyecto de Biomedicina Cima, S.L. | Regulated expression systems |
WO2011122950A1 (en) | 2010-04-01 | 2011-10-06 | Amsterdam Molecular Therapeutics (Amt) Ip B.V. | Monomeric duplex aav vectors |
WO2011133890A1 (en) | 2010-04-23 | 2011-10-27 | University Of Massachusetts | Cns targeting aav vectors and methods of use thereof |
ES2711256T3 (en) | 2010-04-23 | 2019-04-30 | Univ Florida | Compositions of rAAV-guanylate cyclase and methods to treat congenital amaurosis of Leber 1 (LCA1) |
EP2394667A1 (en) | 2010-06-10 | 2011-12-14 | Laboratorios Del Dr. Esteve, S.A. | Vectors and sequences for the treatment of diseases |
TWI557135B (en) | 2010-11-03 | 2016-11-11 | 介控生化科技公司 | Modified factor ix polypeptides and uses thereof |
EP2500434A1 (en) | 2011-03-12 | 2012-09-19 | Association Institut de Myologie | Capsid-free AAV vectors, compositions, and methods for vector production and gene delivery |
EP2691101A2 (en) | 2011-03-31 | 2014-02-05 | Moderna Therapeutics, Inc. | Delivery and formulation of engineered nucleic acids |
EP4043025A1 (en) | 2011-06-08 | 2022-08-17 | Translate Bio, Inc. | Lipid nanoparticle compositions and methods for mrna delivery |
JP6348064B2 (en) | 2011-11-22 | 2018-06-27 | ザ チルドレンズ ホスピタル オブ フィラデルフィア | Viral vectors for efficient transgene delivery |
US9434928B2 (en) | 2011-11-23 | 2016-09-06 | Nationwide Children's Hospital, Inc. | Recombinant adeno-associated virus delivery of alpha-sarcoglycan polynucleotides |
KR20140102759A (en) | 2011-12-16 | 2014-08-22 | 모더나 세라퓨틱스, 인코포레이티드 | Modified nucleoside, nucleotide, and nucleic acid compositions |
JP6383666B2 (en) | 2012-02-15 | 2018-08-29 | バイオベラティブ セラピューティクス インコーポレイテッド | Recombinant factor VIII protein |
AU2013221212B2 (en) | 2012-02-17 | 2018-08-09 | The Children's Hospital Of Philadelphia | AAV vector compositions and methods for gene transfer to cells, organs and tissues |
HUE054087T2 (en) | 2012-04-18 | 2021-09-28 | Childrens Hospital Philadelphia | Composition and methods for highly efficient gene transfer using aav capsid variants |
EP2492347A1 (en) | 2012-05-22 | 2012-08-29 | Laboratorios Del. Dr. Esteve, S.A. | Methods for the production of vectors |
GB201213117D0 (en) | 2012-07-24 | 2012-09-05 | Ucl Business Plc | Transgene expression |
WO2014063108A1 (en) | 2012-10-18 | 2014-04-24 | Biogen Idec Ma Inc. | Methods of using a fixed dose of a clotting factor |
WO2014063753A1 (en) | 2012-10-26 | 2014-05-01 | Vrije Universiteit Brussel | Hyper-active factor ix vectors for liver-directed gene therapy of hemophilia 'b' and methods and use thereof |
CA2888931C (en) * | 2012-10-26 | 2023-09-05 | Vrije Universiteit Brussel | Vector for liver-directed gene therapy of hemophilia and methods and use thereof |
WO2014070349A1 (en) | 2012-10-29 | 2014-05-08 | Regents Of The University Of Minnesota | Factor ix variants |
AU2013348029A1 (en) | 2012-11-20 | 2015-07-02 | The University Of North Carolina At Chapel Hill | Methods and compositions for modified factor IX proteins |
WO2014152940A1 (en) | 2013-03-14 | 2014-09-25 | Shire Human Genetic Therapies, Inc. | Mrna therapeutic compositions and use to treat diseases and disorders |
US20140271550A1 (en) | 2013-03-14 | 2014-09-18 | The Trustees Of The University Of Pennsylvania | Constructs and Methods for Delivering Molecules via Viral Vectors with Blunted Innate Immune Responses |
WO2015012924A2 (en) | 2013-04-29 | 2015-01-29 | The Trustees Of The University Of Pennsylvania | Tissue preferential codon modified expression cassettes, vectors containing same, and use thereof |
US20160207978A1 (en) | 2013-05-06 | 2016-07-21 | Cell Machines Inc. | Methods and compositions related to large scale production of proteins |
CN105408486B (en) | 2013-05-21 | 2020-07-14 | 佛罗里达大学研究基金会有限公司 | Capsid-modified RAAV3 vector compositions and uses in gene therapy of human liver cancer |
PT3024498T (en) | 2013-07-22 | 2020-03-06 | Childrens Hospital Philadelphia | Variant aav and compositions, methods and uses for gene transfer to cells, organs and tissues |
EP3044231B1 (en) | 2013-09-12 | 2020-08-05 | BioMarin Pharmaceutical Inc. | Aav vectors comprising a gene encoding factor viii |
WO2015054439A2 (en) | 2013-10-08 | 2015-04-16 | Haplomics, Inc. | Hybrid factor viii polypeptides for use to treat hemophilia a |
DK3068869T3 (en) | 2013-11-15 | 2020-09-07 | Univ Pennsylvania | Compositions for suppression of factor VIII inhibitor formation in patients with haemophilia A |
EP4332839A2 (en) | 2013-12-06 | 2024-03-06 | Bioverativ Therapeutics Inc. | Population pharmacokinetics tools and uses thereof |
EP2881463A1 (en) | 2013-12-09 | 2015-06-10 | DRK-Blutspendedienst Baden-Württemberg-Hessen gGmbH | Factor IX variants with clotting activity in absence of their cofactor and/or with increased F.IX clotting activity and their use for treating bleeding disorders |
KR101591823B1 (en) | 2013-12-27 | 2016-02-04 | 재단법인 목암생명공학연구소 | Expression vector having an improved gene expression level |
WO2015139093A1 (en) | 2014-03-21 | 2015-09-24 | The Sydney Children's Hospitals Network (Randwick And Westmead) (Incorporating The Royal Alexandra Hospital For Children) | Stable gene transfer to proliferating cells |
AU2015250770B2 (en) | 2014-04-25 | 2020-10-01 | Genethon | Treatment of hyperbilirubinemia |
US11008561B2 (en) | 2014-06-30 | 2021-05-18 | Bioverativ Therapeutics Inc. | Optimized factor IX gene |
WO2016028872A2 (en) | 2014-08-19 | 2016-02-25 | The Children's Hospital Of Philadelphia | Compositions and methods for modulating factor ix function |
US10973931B2 (en) | 2014-09-16 | 2021-04-13 | Universitat Autònoma De Barcelona | Adeno-associated viral vectors for the gene therapy of metabolic diseases |
US20160122739A1 (en) | 2014-10-31 | 2016-05-05 | Wisconsin Alumni Research Foundation | Factor ix variants and methods of use therefor |
WO2016073837A1 (en) | 2014-11-07 | 2016-05-12 | The University Of North Carolina At Chapel Hill | Methods and compositions for modified factor ix proteins |
GB201420139D0 (en) | 2014-11-12 | 2014-12-24 | Ucl Business Plc | Factor IX gene therapy |
CA2975734A1 (en) | 2015-02-06 | 2016-08-11 | The University Of North Carolina At Chapel Hill | Optimized human clotting factor viii gene expression cassettes and their use |
US11007280B2 (en) | 2015-03-17 | 2021-05-18 | Vrije Universiteit Brussel | Optimized liver-specific expression systems for FVIII and FIX |
EP3283126B1 (en) | 2015-04-16 | 2019-11-06 | Emory University | Recombinant promoters and vectors for protein expression in liver and use thereof |
US11104917B2 (en) | 2015-05-08 | 2021-08-31 | Children's Medical Research Institute | Promoters for expression of heterologous genes |
GB201508026D0 (en) | 2015-05-11 | 2015-06-24 | Ucl Business Plc | Capsid |
GB201508025D0 (en) | 2015-05-11 | 2015-06-24 | Ucl Business Plc | Fabry disease gene therapy |
CA2990193A1 (en) | 2015-06-23 | 2016-12-29 | The Children's Hospital Of Philadelphia | Modified factor ix, and compositions, methods and uses for gene transfer to cells, organs and tissues |
CN108024544B (en) | 2015-07-13 | 2022-04-29 | 桑格摩生物治疗股份有限公司 | Delivery methods and compositions for nuclease-mediated genome engineering |
BR112018002150A2 (en) | 2015-08-03 | 2018-09-18 | Bioverativ Therapeutics Inc | factor ix fusion proteins and methods of manufacturing and using them |
CA2992511A1 (en) | 2015-08-03 | 2017-02-09 | Myodopa Limited | Systemic synthesis and regulation of l-dopa |
WO2017070167A1 (en) | 2015-10-20 | 2017-04-27 | The University Of North Carolina At Chapel Hill | Methods and compositions for modified factor ix fusion proteins |
CN108473976B (en) | 2015-10-28 | 2022-07-19 | 桑格摩生物治疗股份有限公司 | Liver-specific constructs, factor VIII expression cassettes, and methods of use thereof |
AU2016362317B2 (en) | 2015-12-01 | 2023-03-16 | Spark Therapeutics, Inc. | Scalable methods for producing recombinant Adeno-Associated Viral (AAV) vector in serum-free suspension cell culture system suitable for clinical use |
CN116732066A (en) | 2016-10-14 | 2023-09-12 | 四川至善唯新生物科技有限公司 | Preparation and application of high-activity blood coagulation factor IX mutant, recombinant protein and fusion protein |
EP3624858A4 (en) | 2017-05-19 | 2021-06-23 | Encoded Therapeutics, Inc. | High activity regulatory elements |
US10842853B2 (en) | 2017-05-22 | 2020-11-24 | Baxalta Incorporated | Viral vectors encoding recombinant fix with increased expression for gene therapy of hemophilia B |
KR102625470B1 (en) | 2017-05-31 | 2024-01-16 | 더 유니버시티 오브 노쓰 캐롤라이나 엣 채플 힐 | Optimized human coagulation factor IX gene expression cassette and use thereof |
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WO2016075473A3 (en) | 2016-08-18 |
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