Factor VIII (FVIII) gene therapy methods
A technology of FVIII and factor, applied in the direction of gene therapy, factor VII, chemical instruments and methods, etc., which can solve the problems of dose limitation
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Embodiment 1
[0303] CpG-reduced Factor VIII DNA sequences and certain vector constructs, plasmid constructs and AAV vector producing cell lines.
[0304] Eighteen different FVIII-encoding reduced CpG nucleic acid variants (SEQ ID NO: 1-18) were generated and evaluated in expression assays. A human FVIII cDNA construct with reduced CpG was generated with a mutant transthyretin (TTRmut) promoter (SEQ ID NO: 22).
[0305] The AAV-SPK-8011 expression cassette has a CpG-reduced FVIII-X07 nucleic acid sequence and a LK03 capsid for packaging. The LK03 capsid has substantial homology to AAV3, a non-pathogenic, naturally replication-defective, single-stranded DNA virus.
[0306] Packaging plasmid pLK03 is a 7,484 bp plasmid construct carrying the AAV2Rep and AAV-LK03Cap genes under the control of the AAV2p5 promoter, a bacterial origin of replication, and a gene that confers resistance to kanamycin in bacterial cells. In this construct, the p5rep promoter has been moved to the 3' end of the cap ...
Embodiment 2
[0320] AAV-SPK-8005 and AAV-SPK-8011 (LK03 capsid, FVIII-X07 (SEQ ID NO:7)) vectors were evaluated in non-human primates (NHP).
[0321]The FVIII transgene construct packaged into an adeno-associated virus (AAV) vector was delivered to non-human primates (NHP). A pilot study (Pilot study) and a GLP study were conducted.
[0322] Briefly, a dose-ranging study was performed in male cynomolgus monkeys given a single intravenous infusion of AAV-SPK-8005 or AAV-SPK-8011 (LK03 capsid). Expression of hFVIII was assessed over 8 weeks. Animal groups and dose levels (pilot studies) for each vehicle as figure 1 shown.
[0323] NHPs receive intravenous fluids via the saphenous vein using a calibrated infusion pump over approximately 30 minutes. Rhesus monkeys were prescreened for neutralizing antibodies against the AAV capsid. All treated animals were initially determined to have titers <1:3 prior to vehicle administration. This was done to ensure successful hepatic transduction, as...
Embodiment 3
[0340] Biodistribution of AAV-LK03 capsids in non-human primates (NHP).
[0341] The biodistribution of the AAV-LK03 capsid in non-human primates was assessed in a non-GLP study. Intravenous injection of an AAV-LK03 encapsidated vector encoding human coagulation factor IX (AAV-LK03-hFIX) revealed that the two main target tissues were the liver and spleen ( Figure 9 ). Spleen tropism is not a unique feature of AAV-LK03. For example, the AAV5 capsid, which has been used in several liver-directed gene therapy trials (e.g., NCT02396342, NCT02082860, NCT02576795), has a strong safety record, and its efficacy in targeting the spleen is comparable to that targeting the liver in nonhuman primates. Equal or higher efficacy (Paneda et al., 2013). The SPK-8011 expression cassette uses the mouse transthyretin or TTR promoters which are believed to be liver specific (Costa, 1991). To further support the liver specificity of the promoter, PCR-based expression analysis determined the ve...
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