Safe and efficient transduction of the liver after peripheral vein infusion of self-complementary AAV vector results in stable therapeutic expression of human FIX in nonhuman primates by Amit C. Nathwani, John T. Gray, Jenny McIntosh, Catherine Y. C. Ng, Junfang Zhou, Yunyu Spence, Melanie Cochrane, Elaine Gray, Edward G. D. Tuddenham, and Andrew M. Davidoff Blood Volume 109(4):1414-1421 February 15, 2007 ©2007 by American Society of Hematology

Plasma clearance of vector and human FIX expression after peripheral vein administration of scAAV2/8 in macaques. Plasma clearance of vector and human FIX expression after peripheral vein administration of scAAV2/8 in macaques. (A) Clearance of the vector from rhesus plasma was determined using a Q-PCR assay on samples collected on days 1, 3, and 7 after peripheral vein administration of scAAV2/8 (□) or scAAV2/5 (▪) vector. Standards consisting of serial dilutions of scAAV2/8-LP1-hFIXco in rhesus plasma were used to define the sensitivity of the assay. Results are expressed as mean transgene copy number per microgram of plasma derived from the 3 animals in each of the serotype 5 and 8 cohorts. (B) Human FIX concentration in rhesus plasma was determined at the indicated time points after administration of 1 × 1012 vg/kg (M5-sc, ○; M6-sc, □; and M7-sc, ▵) serotype 8–pseudotyped scAAV-LP1-hFIXco into the peripheral vein of 3 rhesus macaques. Each sample was independently evaluated on at least 3 separate occasions, and the results are depicted as an average together with the standard error of the mean. Amit C. Nathwani et al. Blood 2007;109:1414-1421 ©2007 by American Society of Hematology

Analysis of the functional activity of hFIX expressed in macaques. Analysis of the functional activity of hFIX expressed in macaques. Schematic of the functional assay, which relies on the ability of the polyclonal rhesus anti-hFIX antibodies to selectively capture hFIX in rhesus plasma, which is then activated by activated factor XI. This was incubated with factor X (FX) in the presence of cofactor (FVIIIa) and phospholipids to generate activated FX (FXa). The amount of FXa is quantitated using chromogenic substrate S2765. (B) A typical standard curve obtained with our functional immunocapture assay demonstrating a relatively linear range for detection of human FIX over 1% to 50% of normal levels using dilutions of human NPP in naive rhesus plasma. (C) Western blot after affinity purification of equivalent amounts of rhesus plasma; 10% SDS-PAGE of affinity-purified hFIX. Lane 1, fresh frozen plasma (FFP) as a positive control; lane 2, sample from monkey M2-sc that received vector into the portal circulation; lane 3, sample from monkey M5-sc that received vector into the systemic circulation; lanes 4 to 6, naive rhesus plasma from 3 animals that had not been transduced with AAV vectors; lane 7 represents 2 mU of affinity-purified hFIX (Replenine) as an additional positive control. Amit C. Nathwani et al. Blood 2007;109:1414-1421 ©2007 by American Society of Hematology

Biodistribution of AAV vector following mesenteric and peripheral vein administration of scAAV2/8-LP1-hFIXco. Biodistribution of AAV vector following mesenteric and peripheral vein administration of scAAV2/8-LP1-hFIXco. (A) Results of semiquantitative PCR analysis in which 1 μg genomic DNA, isolated from the indicated organs at 4 weeks after administration of 1 × 1012 vg/kg scAAV2/8 particles via the mesenteric (top 2 panels) or peripheral venous route, was subjected to PCR using primers unique to hFIXco and designed to amplify a 617 bp product. Integrity of the DNA was determined by amplifying a 604 bp region of the rhesus β-actin gene and is shown at the bottom of each panel. (B) Q-PCR reactions were performed in duplicate to quantitate transgene copy number in each organ after peripheral (⊡, n = 3) and mesenteric (□, n = 2) vein administration of scAAV2/8. The results are represented as vector copy number per diploid genome together with standard errors of mean. Amit C. Nathwani et al. Blood 2007;109:1414-1421 ©2007 by American Society of Hematology

Transduction of rhesus macaques following peripheral vein administration of scAAV2/5. Transduction of rhesus macaques following peripheral vein administration of scAAV2/5. (A) Human FIX concentration in rhesus plasma was determined at the indicated time points after peripheral vein administration of 1 × 1012 vg/kg scAAV vector pseudotyped with serotype 5 capsid (M3-sc, ⋄; M8-sc, □; M9-sc, ○) into 3 rhesus macaques with moderate to high titers of anti-AAV8 antibodies. (B) Human FIX expression in rhesus plasma was determined at the indicated time points after mesenteric vein administration of 1 × 1012 vg/kg scAAV vector pseudotyped with serotype 5 capsid (328, ▿) of a macaque with high titers of anti-AAV8 antibody. Extended expression profile of a macaque previously transduced with scAAV2/5-LP1-hFIXco (M4-sc, □) is shown for comparison. Each sample was independently evaluated on at least 3 separate occasions, and the results are depicted as an average together with the standard error of the mean. Amit C. Nathwani et al. Blood 2007;109:1414-1421 ©2007 by American Society of Hematology

Biodistribution of scAAV2/5-LP1-hFIXco following peripheral vein administration of scAAV2/5. Biodistribution of scAAV2/5-LP1-hFIXco following peripheral vein administration of scAAV2/5. (A) One microgram of genomic DNA, isolated from the indicated organs 4 weeks after peripheral vein administration of 1 × 1012 vg/kg scAAV2/5 particles in M8-sc (top set of panels) and M9-sc (bottom set of panels), was subjected to PCR using primers unique to hFIXco designed to amplify a 617 bp product. Integrity of the DNA was determined by amplifying a 604 bp region of the rhesus β-actin gene and is shown at the bottom of these panels. The middle panel is RT-PCR analysis of RNA extracted from the organs of M8-sc. Integrity of the RNA was determined by amplifying a 295 bp region of the rhesus GAPDH gene and is shown at the bottom of the panel. (B) Q-PCR reactions were performed in duplicate on genomic DNA to establish transgene copy number in each organ after peripheral (⊡, n = 3) and mesenteric (□, n = 2) vein administration of scAAV2/5. The results are represented as vector copy number per diploid genome together with standard errors of mean. Amit C. Nathwani et al. Blood 2007;109:1414-1421 ©2007 by American Society of Hematology

Humoral immune response after mesenteric or peripheral vein administration of scAAV vector. Humoral immune response after mesenteric or peripheral vein administration of scAAV vector. (A) Plasma obtained from macaques after peripheral (M5-sc, ○; M6-sc, □; M7-sc, ▵) or mesenteric vein (M1-sc, broken line and •; M2-sc, broken line and ♦; see Nathwani et al1) administration of scAAV2/8-LP1-hFIXco was analyzed for the presence of AAV8-specific IgG by ELISA (left-hand panel). AAV5-specific IgG titers after peripheral (M8-sc, □; M9-sc, ○) or mesenteric vein (328, broken line and ▾; M4-sc, broken line and ▪) administration of scAAV2/5-LP1-hFIXco are shown in the right-hand panel. (B) Specific isotype profiles of the humoral immune response after administration of scAAV2/8-LP1-hFIXco and scAAV2/5-LP1-hFIXco, respectively (IgM, □; IgG1, ○; IgG2, ▵; IgG4, ⋄). Amit C. Nathwani et al. Blood 2007;109:1414-1421 ©2007 by American Society of Hematology