A Novel Human Factor IX Expression Cassette Packaged as Self-Complementary DNA Dimers in Adeno-Associated Virus Vectors Results in Dramatically Improved Liver Transduction, Significantly Improving Prospects for Hemophilia B Gene Therapy.

Current approaches for gene therapy of hemophilia B (HB) are focused on liver targeted delivery of recombinant adeno-associated virus vectors (rAAV) encoding human Factor IX (hFIX). A major rate limiting step to efficient transduction of the liver is the need to convert the single stranded rAAV genome to a double stranded transcriptionally active form by the host cell. Recently described self-complementary AAV (scAAV) vectors, which can package AAV transgenes as DNA dimers when they are half the size of the wild type genome, bypass the need for second strand synthesis. Their substantially smaller packaging capacity has, however, limited their use for HB gene therapy. We have overcome this obstacle by designing a mini-human FIX expression cassette that is efficiently packaged as self-complementary DNA dimers in rAAV vectors. The key aspects of this novel expression cassette include a truncated regulatory element (LP1) consisting of the critical domains of human apolipoprotein E/C-I gene locus control region and the α1-antitrypsin (hAAT) promoter, and very small intron and polyadenylation elements from SV40. The hFIX 3′untranslated region has been deleted and the coding sequence was re-constructed (hFIXco) using a subset of codons most frequently found in highly expressed eukaryotic genes. The resulting expression cassette, which spanned 2.15 kbp, was cloned into an AAV-2 vector in which the right terminal resolution site had been deleted. Encapsidation with AAV-8 capsid proteins produced the scAAV-2/8 LP1-hFIXco vector in yields comparable to single stranded rAAV. Hirt analysis indicated that all vector genomes in murine liver after tail vein administration existed in a double stranded conformation. Stable plasma hFIX levels at 50% of physiologic (2867 ± 501 ng/ml) were achieved after tail vein administration of 2 x 109 scAAV-2/8 LP1-hFIXco vector/mouse. This is >500 fold higher than levels achieved with standard single stranded rAAV-2 vectors and >10 fold higher than levels achieved with a comparable single stranded vector pseudotyped with AAV-8 capsid proteins, confirming the superiority of scAAV vectors. Further increases in scAAV-2/8 LP1-hFIXco vector dose to 1 x 1011 particles/mouse resulted in a proportionately linear increase in stable plasma hFIX levels to over 100 μg/ml without any adverse toxicity. Importantly, transgene expression mediated by the LP1 promoter/enhancer appears to be strictly restricted to the liver. Evaluation of the efficacy and safety of scAAV-2/8 LP1-hFIXco in rhesus macaques is ongoing. However, based on the murine studies, our novel self complementary hFIX expressing vector offers a unique opportunity to mediate therapeutic gene transfer in humans without the need for higher vector doses. This has important safety implications and will therefore substantially improve the prospects of hemophilia gene therapy.