Hanley, JE; (2014) The use of matrix attachment regions to enhance the in-vivo potency of rAAV vectors. Doctoral thesis , UCL (University College London).

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Abstract

Adeno-associated virus vector encoding codon optimised human factor IX (AAV-LP1-hFIXco) has demonstrated great promise for the treatment of patients with severe haemophilia B. However, in some patients treated with a high vector dose, hepatocellular toxicity was observed. To improve AAV vector potency, various scaffold/matrix attachment regions (S/MARs) were cloned at the 3’ end of a modified single-stranded (ss) AAV-LP1-hFIXco expression cassette. In a head to head comparison, a vector containing a S/MAR element from the human hypoxanthine-guanine phosphoribosyl-transferase gene in the forward orientation (ssAAV-LP1-hFIXco-HPRT-F) was found to mediate the highest levels of hFIX expression in mice. In comparison to animals transduced with a control vector containing no S/MAR, the ssAAV-LP1-hFIXco-HPRT-F transduced cohort expressed hFIX at 28-fold higher levels. This trend was reproducible in rhesus macaques where 10-fold higher FIX levels were observed following transduction with ssAAV-LP1-hFIXco-HPRT-F as compared to delivery of ssAAV-LP1-hFIXco-control vector. Through a deletion analysis, short regions from the IFNβ and HPRT S/MARs with potent enhancer activity were identified. This allowed for the in-silico elicitation of motifs with a potential role in S/MAR function and also minimised the space occupied by S/MARs within our AAV expression cassette. When cloned into a self-complementary (sc) AAV-LP1-hFIXco expression cassette, the 130bp region from the HPRT S/MAR (fragment 2b) was sufficient to enhance FIX levels in mice by 35-fold over that observed with a control self-complementary vector. Mechanistic studies showed that S/MAR elements enhanced AAV transgene expression by reducing heterochromatin marks (H3K9me2 and HP1α) in the promoter region, resulting in an increase in FIX mRNA levels by up to 20-fold. S/MARs therefore provide a novel inbuilt process for enhancing AAV mediated transgene expression by preventing epigenetic silencing of the provirus. As such, S/MARs offer the possibility to improve gene transfer to humans through using lower and potentially safer doses of AAV.

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