Volume 19, Issue 5, Pages 876-885 (May 2011) Long-term Safety and Efficacy Following Systemic Administration of a Self- complementary AAV Vector Encoding Human FIX Pseudotyped With Serotype 5 and 8 Capsid Proteins  Amit C Nathwani, Cecilia Rosales, Jenny McIntosh, Ghasem Rastegarlari, Devhrut Nathwani, Deepak Raj, Sushmita Nawathe, Simon N Waddington, Roderick Bronson, Scott Jackson, Robert E Donahue, Katherine A High, Federico Mingozzi, Catherine YC Ng, Junfang Zhou, Yunyu Spence, M Beth McCarville, Marc Valentine, James Allay, John Coleman, Susan Sleep, John T Gray, Arthur W Nienhuis, Andrew M Davidoff  Molecular Therapy  Volume 19, Issue 5, Pages 876-885 (May 2011) DOI: 10.1038/mt.2010.274 Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Clearance of self-complementary AAV vector (scAAV)2/8-LP1-hFIXco vector after peripheral vein administration of 2 × 1012 pcr-vector genomes (vg)/kg. Clearance of the vector from rhesus plasma, urine, saliva, and stool was determined using a quantitative PCR (qPCR) assay on samples collected following peripheral vein administration of 2 × 1012 pcr-vg/kg scAAV2/8-LP1-hFIXco. 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 (vg)/ml ± SE of sample obtained from three animals in this dose cohort and indicate that the vector genomes have cleared from the body fluids by day 10. Molecular Therapy 2011 19, 876-885DOI: (10.1038/mt.2010.274) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Expression of human factor IX (hFIX) in rhesus macaques transduced with different doses of adeno-associated viral (AAV) vectors. Plasma hFIX levels (mean ± SEM) measured by enzyme-linked immunosorbent assay (ELISA) at the stated time points following peripheral vein administration of scAAV-LP1-hFIX of (a) 2 × 1012 pcr-vg/kg, (b) 2 × 1011 pcr-vg/kg, (c) 6 × 1010 pcr-vg/kg, and (d) 2 × 1010 pcr-vg/kg. (e) The relationship between vector dose and plasma hFIX levels and scAAV transgene copy number in the liver at 4 weeks after gene transfer. Molecular Therapy 2011 19, 876-885DOI: (10.1038/mt.2010.274) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Biodistribution of vector following peripheral vein administration of 2 × 1012 pcr-vg/kg scAAV2/8-LP1-hFIXco. Results of quantitative PCR (qPCR) analysis of genomic DNA, isolated from the indicated organs at 8 weeks after administration of 2 × 1012 pcr-vg/kg of scAAV2/8 particles via the peripheral venous route using primers unique to hFIXco. Shown is transgene copy number per diploid genome ± SE corrected for variation in loading and amplification efficiency using GAPDH primers. BM, bone marrow. Molecular Therapy 2011 19, 876-885DOI: (10.1038/mt.2010.274) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Humoral immune response after peripheral vein administration of self-complementary AAV vector (scAAV) vector. Plasma obtained from macaques after peripheral administration of scAAV2/8-LP1-hFIXco at the stated doses and at different time points after vector administration was analyzed for the presence of adeno-associated virus (AAV)8-specific immunoglobulin G (IgG) by enzyme-linked immunosorbent assay (ELISA) ± SE. Assays were performed in triplicate. Molecular Therapy 2011 19, 876-885DOI: (10.1038/mt.2010.274) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Long-term expression of human factor IX (hFIX) following a single bolus infusion of research grade self-complementary adeno-associated viral vector (scAAV)-LP1-hFIXco. (a) Plasma human FIX (hFIX) levels (mean ± SEM) measured by enzyme-linked immunosorbent assay (ELISA) at the stated time points following a single bolus infusion of scAAV-LP1-hFIXco into the peripheral vein using serotype 8 pseudotyped vector (arrows show points when liver biopsies were obtained). Macaques in these studies have been described previously.8,11 The bottom panel shows Southern blot analysis of genomic DNA extracted from the liver obtained at the stated time points after peripheral vein administration of scAAV2/8-LP1-hFIXco and digested with BsrDI followed by hybridization with an hFIXco-specific probe at the stated time. Arrow shows the expected ∼1.5 kb hybridization band. (b) The relationship between plasma hFIX levels and transgene copy number in the liver. Plasma hFIX levels (mean ± SEM) following a single bolus infusion of scAAV-LP1-hFIXco into the (c) mesenteric vein using serotype 8 pseudotyped vector, and (d) peripheral vein using serotype 5 pseudotyped vector. Gray arrows with “R” in Figure 5c,d denote administration of the combination of rituximab (325 mg/m2) and cyclophosphamide (250 mg/m2) as a bolus weekly infusion for a period of 4 weeks. Molecular Therapy 2011 19, 876-885DOI: (10.1038/mt.2010.274) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Expression of human factor IX (hFIX) following in-utero delivery of self-complementary adeno-associated viral vector (scAAV)2/8-LP1-hFIXco in mice. Correlation between mean body mass (weight in grams) and plasma human FIX (hFIX) levels (mean ± SEM) measured by enzyme-linked immunosorbent assay (ELISA) at the stated time points following a single in-utero administration of scAAV2/8-LP1-hFIXco. Molecular Therapy 2011 19, 876-885DOI: (10.1038/mt.2010.274) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions