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ADVANCES IN GENE THERAPY FOR HEMATOLOGIC DISORDERS cells of mice with β-thalassemia intermedia (Blood, First Edition, Oct 2002) This vector utilized 1.7 kb of enhancer sequences from hypersensitive site elements 2, 3, and of the β-globin locus control region (LCR) linked to a 130 bp β-globin promoter to drive expression of the Aγ-globin gene Using this vector, 25% of mice transplanted with genetically modified cells demonstrated pancellular γ-globin expression and correction of anemia as reflected by a 2.5 g/dL increase in hemoglobin (Hb) However, the majority of transplanted mice showed heterocellular expression and only a g/dL increase in Hb, despite having an average vector copy (VC) number similar to the first group (VC of 2.1 vs 2.4) Analysis of γ-globin expression in unique, clonal CFU-S erythroid cells derived from these animals verified that detrimental chromosomal position effects often compromised expression To improve expression, we developed a second-generation γ-globin lentiviral vector containing a 3.5 kb LCR configuration previously shown to direct significant expression at “non-permissive” chromosomal locations in erythroid cell lines (Molete et al., Mol Cell Biol 21:2969) Despite preemptive ablation of five undesirable polyadenylation signal sequences (AATAAA) contained in the new vector, the titer remained suboptimal (3.8 ± 1.8 x 105 TU/ml) Northern and 3’RACE analysis of producer cell RNA showed persistent premature polyadenylation occurring in a restricted region within the vector, despite the lack of consensus AATAAA sequences Deletion of this region rescued vector titer (1.8 ± 0.5 x 106 TU/ml) A direct comparison of the therapeutic efficacy of this modified 3.2 kb LCR vector and the 1.7 kb LCR vector was then performed Five of six mice transplanted with βthalassemic stem cells transduced with the 3.2 kb LCR vector showed therapeutic levels of chimeric mα2hγ2 Hb tetramers (HbF), ranging from 17-33% of total Hb (mean 26% ± 2% ) In contrast, only two of five animals transplanted with cells transduced with the 1.7 kb vector had HbF levels above 10% (overall mean 8% ± 2%) The high level of HbF in the animals receiving cells transduced with the larger vector was obtained with a VC number of 1.3 ± 0.3 and resulted in correction of anemia with a mean Hb of 11.2 ± 0.3 (n=6), 2.6 g/dL higher than animals transplanted with mock-transduced cells (8.6 ± 0.4, n=5) In contrast, phenotypic improvement was more modest in the 1.7 kb LCR group which displayed a Hb level of 10.3 ± 0.3 (n=5) despite a VC number of 2.0 ± 0.3 The output of HbF and the increase in total Hb level per VC were also much greater in the 3.2 kb LCR vector group (18% ± 3% HbF per VC and 2.0 g/dL Hb per VC) than in the smaller LCR vector group (4% ± 0.4% HbF per VC and 0.9 g/dL Hb per VC) We conclude that inclusion of extended LCR sequences in γ-globin vectors is feasible after elimination of undesirable polyadenylation sites and results in significant augmentation of therapeutic efficacy in the context of more desirable lower VC numbers VECTOR GENOME BIOLOGY 400 Diversity of Latent AAV Genomes in NonHuman Primate and Human Tissues Guangping Gao,1,3 Mauricio R Alvira,1,3 You Lu,1,3 Luk H Vandenberghe,1,3 Suryanarayan Somanathan,1,3 Lihui Lai,2 Maurice J Duplantis,2 Bruce A Bunnell,2 James M Wilson.1,3 University of Pennsylvania School of Medicine, Philadelphia, PA; Tulane University Health Sciences Center, New Orleans, LA; The Wistar Institute, Philadelphia, PA We recently reported isolation and vector development of two novel AAV serotypes, named AAV7 and 8, from rhesus macaque tissues Vectors based on AAV7 and have demonstrated superior performance in several animal models In our continued effort to isolate more potent AAV vectors, a total of 265 AAV capsid clones were generated from 260 tissues of 29 NHP animals of species and 206 NHP blood samples of species Among those 265 clones, 57 S158 clones were fully sequenced and compared to the published AAV sequences This analysis revealed a great diversity of latent AAV genomes in NHP populations These 57 clones were derived from 17 different animals of species, among which at least 40 novel AAV capsid sequences were considered non-redundant based on the finding of amino acid differences from one another The structural relationship between these non-redundant AAV Cap sequences and previously reported AAV genomes were determined using the Splits Tree program with implementation of the method of split decomposition The endogenous viruses are widely distributed throughout NHP tissues with the most abundant in liver, heart, lung, spleen and lymph nodes Further analysis of 15 clones isolated from the lymph nodes of a single animal suggested that in vivo homologous recombination between AAV genomes contribute to AAV diversity More recently, we examined latent AAV genomes in human tissues Of 65 tissues from 29 individuals analyzed so far, 10 samples from different tissues were positive by PCR Among those positive tissues, liver stands out, resulting in strong amplifications in out of 12 liver samples Initial analysis on some clones from two human livers indicated that novel AAV sequences present in human livers are variants of human AAV serotype Some degree of sequence diversity was also observed among the clones from the same liver Interestingly, hybrid genomes consisting of sequences from two human AAV serotypes (2 and 3) were identified, implying that in vivo homologous recombination of AAV genomes in human tissues could be the same mechanism as in NHP that leads to evolution and diversity of AAV genomes James M Wilson holds equity in Targeted Genetics, Corp 401 Serial Real-Time Luminescence Imaging Profiles Differential Kinetics and Biodistibution of Transgene Expression from AAV Serotype 1-5 Vectors Following Adult and Neonatal Gene Transfer Paul E Monahan,1 Joseph E Rabinowitz,1 Joseph R Elia,1 Yi Tang,2 Vasilis Ntziachristo,2 Brian S Sorg,3 Mark W Dewhirst,3 Ralph Weissleder,2 R Jude Samulski.1 Gene Therapy Center, University of North Carolina, Chapel Hill, NC; 2Radiology, Massachusetts General Hospital, Charlestown, MA; 3Radiation Oncology, Duke University Medical Center, Durham, NC Definition of the timing and magnitude of vector expression (generally measured by serial in vivo sampling) and vector biodistribution (by post-mortem screening of tissues and fluids) are essential for the design of safe, efficacious gene therapy Biodistribution of transgene expression from adeno-associated virus (AAV) serotype vectors has been examined, but newer alternativeserotype AAV vectors have undefined transduction patterns and kinetics We performed simultaneous in vivo monitoring of expression and biodistribution of firefly luciferase (luc) marker gene delivered in AAV serotype 1-5 vectors A cooled charged coupled device (CCCD) camera was used to quantify non-invasively low level bioluminescent photons transmitted from intact animals expressing luc Adult mice (n=3-5/group) were injected with AAV serotypes 1, 2, 3, or 5/CMV promoter/luc (1 x 1011 vector genomes/ animal) intramuscularly (I.M.) or AAV2-5 via the portal vein Wholebody imaging was performed serially beginning as early as day post injection (PI) and continued to 100 days PI Additionally, neonatal Balb/C mice ( 100-fold higher expression than AAV2 and AAV4 in muscle; expression after I.M delivery localized exclusively to the injected limb with all serotypes Following adult portal vein delivery, AAV2-mediated luminescence was evident at low levels in the first week, but peaked in the 2nd month post-treatment Peak expression from AAV3 was similar to AAV2 and greater than AAV4 and AAV5 Following neonatal intravenous injection AAV3 led to minimal expression in the neonatal period While the site of intravenous injection itself was effectively transduced with all serotypes at this age, the vectors otherwise had serotype-distinctive tissue expression profiles AAV1 expressed primarily in skeletal muscle and cardiac muscle Skeletal muscle expression was 20 to 50-fold greater/mcg of tissue protein using AAV1 than other serotypes AAV2 led to high total luminescence in liver Markedly higher local expression at the site of systemic injection was seen in neonates (temporal vein) than adults (portal vein) This and additional serotype- and tissue-specific bioluminescence differences suggest physiologic differences between neonates and adults affect transgene expression, which will inform design and monitoring of tissue-targeted gene therapy strategies 402 AAV Serotype Vectors Preferentially Integrate into Active Genes in Mice Hiroyuki Nakai,1 Eugenio Montini,2 Sally Fuess,1 Theresa A Storm,1 Markus Grompe,2 Mark A Kay.1 Departments of Pediatrics and Genetics, Stanford University School of Medicine, Stanford, CA; 2Departments of Molecular and Medical Genetics and Pediatrics, Oregon Health & Science University, Portland, OR Recombinant AAV serotype (rAAV2) is a promising vector for gene therapy because it can achieve long-term stable transgene expression in animals In the liver, although stable transgene expression primarily results from non-integrated vector genomes, a series of mouse experiments have demonstrated that vector genomes integrate into chromosomes in hepatocytes at a low frequency Despite the low integration efficiency, recent reports of retroviral insertional mutagenesis in mice and human subjects have raised concerns about the potential for rAAV2-mediated insertional mutagenesis However, host chromosomal effects of rAAV2 vector integration and target site selection in quiescent somatic cells in animal tissues are not known due to the lack of an efficient system that allows for isolation of the infrequently integrated proviruses from the large number of extrachromosomal vector genomes in transduced tissues To this end, we utilized an in vivo hepatocyte selection system based on a hereditary tyrosinemia type I (HTI) mouse model HTI is an inherited fatal metabolic hepatorenal disease caused by deficiency of fumarylacetoacetate hydrolase (FAH) Because there is a selective repopulation of genetically modified FAH-positive hepatocytes in HTI mouse livers, it allows for in vivo clonal selection of hepatocytes with integrated rAAV2 vector genomes expressing FAH With injection of a FAH-expressing rAAV2 vector carrying the ampicillin resistance gene and plasmid ori, into HTI mouse livers followed by in vivo selection, we isolated 14 whole proviral vector genomes with both junction sequences, with a plasmid rescue technique Eleven proviral genomes were monomers while complicated structures were found in cases Viral sequences were frequently deleted at the termini We identified deletions of cellular genomes at all the integration sites (14 of 14 integrations), ranging from bp to ~0.3 kb in most cases, while a 2.1 kb deletion occurred in a single integration event We found no significant homologies between vector and cellular DNA, but frequently found microhomologies around the integration sites With Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy the information from a total of 29 integration sites identified in rAAV2-injected mouse livers without selection (15 integrations previously published) and in vivo selected rAAV2-transduced hepatocytes (14 integrations in the present study), we began to elucidate how rAAV2 vectors might select target sites for integration Although the integration sites seemed to be randomly distributed on mouse chromosomes, rAAV2 integration was highly favored to occur within genes (both exons and introns), regardless of whether or not the vector-cellular DNA junctions were isolated after selective pressure (12 of 15, under non selection; of 14, under selective pressure; overall frequency, 21 of 29, 72%) Importantly, web-based public databases and our RT-PCR analysis confirmed that 19 /19 target genes analyzed were expressed in the liver This is the first report on host chromosomal effects of rAAV2 integration and target site selection in quiescent somatic cells in animals, and provides insights into the nature of rAAV2 vector integration, which will be useful in establishing the risk of insertional mutagenesis 403 Targeted Correction of Genomic Single Base-Pair Mutations Using Adeno-Associated Virus under Non-Selective Conditions Xiaoming Liu,1 Ziying Yan,1 Meihui Luo,1 Roman Zak,1 Ryan Driskell,1 Ziyi Li,1 John F Engelhardt.1,2 Anatomy and Cell Biology, The University of IOwa, Iowa City, IA, United States; 2The Center for Gene Therapy of Cystic Fibrosis and Other Genetic Disease, The University of Iowa, Iowa City, IA, United States Recombinant adeno-associated virus (rAAV) has attracted considerable interest as a vector for gene therapy One of the more fascinating, but less-well studied, aspects of this vector includes its ability to direct single base pair changes at sites of vector homology in genomic DNA through a mechanism thought to involve proofreading repair In order to further study this feature of rAAV, we created a non-selectable EGFP-based reporter system in a 293-cell line and transgenic mice This model utilizes an EGFP gene that does not fluorescence due to a single base-pair substitution and provides for a real-time evaluation of gene targeting in living cells without the need for selective pressure Several rAAV vectors containing various segments of the EGFP gene and flanking regions were tested for their ability to correct an integrated mutant EGFP gene and recover fluorescence in the 293-cell line model system None of these vectors produced fluorescent protein in naive cells not containing the EGFP target rAAV virus containing the longest possible length of short end homology to the GFP target sequence (~440bp) demonstrated a remarkable 0.1% efficiency of gene targeting based on FACS analysis of recovery of GFP fluorescence FACS sorting was also used to isolate and expand clonal populations of targeted cells for conclusive identification of gene repair Isolated cell clones were cultured and continuously expressed fluorescent EGFP and sequence confirmation of the single base conversion in the integrated genomic target was confirmed by PCR sequencing These results support previous findings by Russell and colleagues demonstrating the efficacy of rAAV for gene targeting Our data also showed that the efficiency of rAAV mediated gene targeting can be improved by increasing the length of homology between the AAV vector and the target locus and the vector multiplicity of infection However, neither genotoxic stress or proteasome inhibitors enhance the efficiency of the targeting event Since both of these manipulations increase transduction with rAAV, but not uptake of virus, these results suggest that a non-expressing intermediate of the viral genome is responsible for mediating gene conversion events Interestingly, our result demonstrated that enrichment of S-phase cells in the target population, by treatment with thymidine, led to a 3-5 fold higher level of correction and suggests that the phase of the cell cycle may affects gene targeting with rAAV vectors Results studying S159 ... will inform design and monitoring of tissue-targeted gene therapy strategies 4 02 AAV Serotype Vectors Preferentially Integrate into Active Genes in Mice Hiroyuki Nakai,1 Eugenio Montini ,2 Sally... of rAAV2 integration and target site selection in quiescent somatic cells in animals, and provides insights into the nature of rAAV2 vector integration, which will be useful in establishing the... expression from AAV2 and AAV3 was only beginning AAV1 and AAV5 led to > 100-fold higher expression than AAV2 and AAV4 in muscle; expression after I.M delivery localized exclusively to the injected limb