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. 2009 Oct 27;18(1):118–125. doi: 10.1038/mt.2009.246

Systematic Evaluation of AAV Vectors for Liver directed Gene Transfer in Murine Models

Lili Wang 1, Huan Wang 1,2, Peter Bell 1, Robert J McCarter 3, Jianping He 3, Roberto Calcedo 1, Luk H Vandenberghe 1, Hiroki Morizono 3, Mark L Batshaw 3, James M Wilson 1
PMCID: PMC2839210  PMID: 19861950

Abstract

Vectors based on adeno-associated viruses (AAVs) are being evaluated for use in liver-directed gene therapy. Candidates have been preselected on the basis of capsid structure that plays an important role in determining performance profiles. We describe a comprehensive and statistically powered set of mouse studies designed to compare the performance of vectors based on seven novel AAV capsids. The key criteria used to select candidates for successful gene therapy are high level and stable transgene expression in the absence of toxicity. Based on these criteria, the best performing vectors, AAV8, AAVhu.37, and AAVrh.8, will be further evaluated in nonhuman primates (NHPs).

Introduction

The liver plays an important role in most metabolic pathways as well as in the production of many serum proteins. These functions have been the target of genetic manipulation in the treatment of disease through ex vivo and in vivo approaches in preclinical and clinical models.1,2,3 The most practical approach for widespread clinical applications of liver-directed gene therapy, however, is in vivo targeting following systemic administration of the vector. The endothelial fenestrations present in the hepatic microcirculation allow easy access of vectors to hepatocytes following vascular administration, a unique property of liver.

A number of viral and nonviral vectors have been evaluated for in vivo, liver-directed gene transfer, although those based on adeno-associated viruses (AAVs) appear to be the most promising. The most extensive experience has been with vectors based on AAV serotype 2 that show stable transduction of hepatocytes with minimal transgene immune responses in murine models.4 Several limitations of AAV serotype 2 vectors have emerged including low transduction efficiency, high seroprevalence of neutralizing antibodies (NAbs) in humans, and potentially destructive T-cell responses to capsids.3,5,6,7

The repertoire of AAV vectors available for gene transfer has been markedly expanded through the isolation of natural variants and the engineering of capsids.8,9,10 Many of the novel capsids isolated from primate sources have been shown by us and others to have improved properties as vectors including higher transduction, lower seroprevalence, and diminished capsid immune responses.6,11,12,13,14,15 However, due to variations in key experimental details such as dose, vector production method, transgene, animal model, etc., it is difficult to compare many of these studies.

In this study, we have undertaken a systematic evaluation of seven promising novel AAV capsids for liver gene transfer in mice based on a comprehensive panel of relevant biological properties.

Results

A portfolio of 30 AAV capsids was established from a larger family of over 120 novel AAV capsids that we previously isolated from nonhuman primates (NHPs) and human tissues.8,9,10 A subset of these vectors has been analyzed for transgene and capsid T-cell activation, toxicity, biodistribution, and pre-existing immunity in human populations. Based on these pilot studies, we selected seven capsids for a more detailed and systematic analysis as potential vectors for human applications of liver-directed gene therapy, the topic of this study. The rationale for selecting these seven candidates is provided below.

Capsids were selected to represent the overall biodiversity of the larger family of natural AAV isolates. Vectors based on these 30 viruses have been evaluated in a number of different settings for vector performance such as gene transfer in liver, muscle, and lung. As can be seen in Figure 1, the seven candidates span three clades (A, D, and E) as well as two isolates that fall outside of clades. AAV7 and AAV8 were selected based on extensive prior work demonstrating their potential for liver-directed gene therapy including studies in NHPs.16,17,18 AAVrh.64R1 and AAVhu.37 are other members of the clade E family, isolated from a rhesus macaque and human, respectively, that demonstrate high transduction efficiency across a broad spectrum of tissues. AAVrh.8 phylogenetically clusters between AAVs 7 and 8 but is not part of a distinct clade; pilot studies indicate moderate transduction of liver. AAVrh.32.33 is not related to any clade, although it shares similarity to AAV4. Transduction of liver is modest, although pre-existing immunity is almost nonexistent in humans.6 AAV6.2 provides the highest level of liver transduction of any clade A member,10 and, in lung, it is less immunogenic in terms of producing NAb (M. Limberis and J.M. Wilson, unpublished results).

Figure 1.

Figure 1

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Phylogenetic relation of candidate AAVs to other primate AAV capsids considered for gene therapy. Dendrogram of VP1 protein sequences was derived by neighbor-joining algorithm. Candidate capsids are underlined. Scale, evolutionary distance of the number of substitutions per site. AAV, adeno-associated virus.

These candidate vectors were included in a variety of experiments to assess their relative performance according to several relevant criteria (see Table 1): (i) transduction efficiency; (ii) stability of transgene expression; (iii) hepatotoxicity as measured by peak serum transaminases; (iv) liver histopathology; (v) activation of T cells to capsids; (vi) activation of T cells to an antigenic transgene product; (vii) presence of pre-existing NAb in human populations and their impact on inhibiting in vivo transduction in passive transfer studies; and (viii) distribution of vector genomes outside of the liver. The seven vectors were compared to one another with respect to each of these biological properties establishing a relative performance score. The scores for an individual criterion ranging from −5 (least favorable) to +5 (most favorable) were assigned following statistical analysis (see Materials and Methods). The final rank order of vector candidates was established based on a summation of all performance scores.

Table 1.

Scores for AAV candidates based on statistical analysis

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Efficiency and stability of transgene expression were evaluated following intravenous injection of AAVs that express green fluorescent protein (GFP) from the constitutive and ubiquitous CB (i.e., cytomegalovirus-enhanced chicken β-actin) promoter at 1 × 1011 genome copies/mouse. Animals were sacrificed at different time points, and liver tissue was processed for fluorescence microscopy (Figure 2a) and morphometric quantification (Figure 2b). Peak expression was essentially identical for all vectors except AAVrh.32.33 that was about tenfold lower. Over a 120-day period, expression was essentially stable for AAV8 and AAVrh.64R1, significantly reduced for AAV7 and AAVrh.32.33, and slightly reduced for the others (i.e., AAVhu.37 and AAVrh.8 slightly more stable than AAV6.2).

Figure 2.

Figure 2

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AAV-mediated transgene expression in mouse liver. Livers were harvested, sectioned, and photographed at 7, 14, 28, 90, and 120 days after intravenous (i.v.) injection of AAV.CB.EGFP. (a) EGFP expression in liver at day 90 (vector dose at 1 × 1011 GC/mouse). Images representing the median expression level of the group are shown. Bar = 150 µm. (b) Time course of GFP transduction efficiency in mouse liver (vector dose at 1 × 1011 GC/mouse). The intensity of the green fluorescence was quantified with ImageJ. Data are presented as median with 95% CI (confidence interval) for each group (n = 4–6). Dotted line indicates background level from a naive mouse liver. (c) GFP transduction efficiency in mouse liver injected with tenfold lower vector dose (1 × 1010 GC/mouse). AAV, adeno-associated virus; EGFP, enhanced green fluorescent protein; GC, genome copy.

Similar expression studies were performed at tenfold lower doses of vector for all capsids except AAV6.2. Transgene expression as measured by GFP brightness was diminished in proportion to the dose of vector and was generally equivalent across all vectors when measured at day 7. Expression was stable and slightly increased over 28 days for all vectors except AAVrh.32.33 that demonstrated a 3 log reduction to below detection levels (Figure 2c).

Toxicity focused on liver pathology was assessed by measurement of serum transaminases and histopathology. All vectors demonstrated transient increases in transaminases at day 14 that were minor and virtually indistinguishable from one another except AAVrh.64R1 that had slightly higher peak levels and AAVrh.32.33 in which the rise in transaminases was much higher, resembling what is seen with first-generation adenoviral vectors (Figure 3). Histological analysis revealed pathology characterized as portal and lobular inflammation, both of which were scored on a scale of 0–3. Micrographs of representative histological sections are shown in Figure 4a, and the scoring data are presented in Figure 4b. The data segregated into two groups with the least inflammation found in AAV6.2, AAV7, and AAVrh.8, and slightly more inflammation in the remaining four.

Figure 3.

Figure 3

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Transient elevation of liver enzymes in mice following intravenous administration of 1 × 1011 GC of AAV.CB.EGFP. (a) Time course of liver enzyme levels showing peak elevation at day 14. Data are presented as median with 95% confidence interval for each group [n = 30 (pre and day 7), 24 (day 14), 18 (days 28 and 42), and 6 (day 90)]. (b) Elevation of ALT and AST at day 14 for individual mice. AAV, adeno-associated virus; ALT, alanine aminotransferase; AST, aspartate aminotransferase; EGFP, enhanced green fluorescent protein; GC, genome copy.

Figure 4.

Figure 4

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Temporary liver inflammation at day 14 following vector administration. (a) Representative hematoxylin and eosin (H&E) staining of formalin-fixed and paraffin-embedded livers harvested at day 14 after vector injection. Bar = 100 µm. (b) Scores for portal and lobular inflammation in individual mice. Horizontal bar indicates median level of the group (n = 4–6). AAV, adeno-associated virus.

Splenocytes were harvested at multiple time points and evaluated for T-cell responses against capsid (Figure 5a) and GFP (Figure 5b) using an ELISPOT assay measuring the number of cells stimulated to produce IFN-γ. Peak T-cell response to capsid was at day 7, although it was generally low for all vectors except AAV6.2 that produced over 1,000 spots/106 cells. AAVrh.32.33 did not elicit a detectable capsid T-cell response, whereas low-level responses were observed with the remaining vectors. Peak GFP responses were at day 14 and ranged from over 1,000 spots/106 cells for AAVrh.32.33 to 370–530 spots/106 cells for AAVs 8, 6.2, 7, rh.64R1, and hu.37, and to ~200 spots/106 cells for AAVrh.8.

Figure 5.

Figure 5

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T-cell response to AAV capsid and transgene GFP. Splenocytes were isolated 7, 14, 28, and 90 days after intravenous administration of 1 × 1011 GC of AAV.CB.EGFP. For IFN-γ ELISPOT assay, splenocytes from individual animals were stimulated in vitro with the peptides containing the dominant H-2d-restricted CD8 T-cell epitopes of AAV capsid or GFP, respectively. Time course of the T-cell response to (a) AAV capsid and (b) GFP are shown here. Data are presented as median with 95% confidence interval for each group (n = 4–6). AAV, adeno-associated virus; EGFP, enhanced green fluorescent protein; GC, genome copy; SFC, spot forming cell.

A critical aspect of a vector to be administered intravenously is potential inhibition of transduction by NAb from natural infections. Our analyses were based on a screen of human serum samples for NAb using the in vitro transduction inhibition assay and an in vivo passive transfer assay; data from AAV2 were included for reference. Sera from 40 to 100 human volunteers were evaluated for the presence of NAb at a titer of >1:20 (Figure 6a); the exact titer was evaluated for those samples that were positive (Figure 6b). The prevalence of low-level NAb was highest for AAV2 and lowest for AAVrh.32.33, with the rest in a large but indistinguishable group in between. Passive transfer studies were performed by administering pooled human intravenous immunoglobulin (IVIG) to mice before delivering vectors that expressed cFIX made from the candidate capsids (Figure 6c,d). The impact of the IVIG on transduction was assessed by measuring day 28 plasma for the transgene product cFIX by enzyme-linked immunosorbent assay. For each vector, different quantities of IVIG were injected (0, 1.2, 4, 12, and 40 mg) to allow for in vivo titration of the effective neutralizing activity. All vectors demonstrated a dose-dependent inhibition of transgene expression with 12 mg of IVIG as the most informative in terms of differentiating the vectors. Greater than 70% inhibition was noted for AAVs hu.37, 8, and 2 with detectable but less substantial inhibition measured for the rest and least diminution with rh.8. AAV2 was the only vector that showed >90% reduction at a tenfold lower dose of IVIG consistent with its higher NAb activity in the human population. When combining the goodness of fit data for both seroprevalence and passive transfer, most vectors were indistinguishable from one another except AAVs rh.8 and rh.32.33 that showed favorable scores (Table 1; AAV2 was not included in the candidate-selection statistical analysis).

Figure 6.

Figure 6

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Prevalence of neutralizing antibodies against different AAV types in humans evaluated by in vitro NAb assay on human sera and inhibition of in vivo gene transfer by passive transfer of pooled human IVIG. (a) Percentage of human serum samples with NAb titer >1:20. (b) Magnitude of NAb titers for each AAV type. NAb to AAV2 is included as a reference. Number of samples assayed for AAV2, 7, 8, and rh.32.33: n = 100 (data have been published previously6); AAV6.2: n = 50; AAVhu.37, rh.64R1, and rh.8: n = 40. (c) C57BL/6 mice received passive transfer of human IVIG at the indicated amount at 24 and 2 hours before vector administration of 1 × 1011 GC of AAV.LSP.cFIX-W packaged with different AAV capsids. Canine factor IX (cFIX) expression levels in plasma of individual mice at day 28 following vector administration are shown. Horizontal bar indicates median level of the group (n = 4–5). (d) Level of inhibition shown by the ratio of median cFIX levels in mice that received IVIG to the median levels in control mice (0 mg of IVIG). Data are presented as median with 95% confidence interval for each group (n = 4–5). (e) Time course of cFIX expression levels in the plasma of control mice (0 mg of IVIG) by seven candidates. AAV, adeno-associated virus; GC, genome copy; IVIG, intravenous immunoglobulin; NAb, neutralizing antibodies.

The passive transfer studies provided another context to assess relative transduction efficiency in the absence of NAb to AAV by comparing peak factor IX expression at day 28 (Figure 6e). Expression as measured by plasma factor IX levels were in the same general range, differing no more than threefold across all seven candidates with AAVrh.32.33 being the lowest. Expression was relatively stable over 83 days including that obtained with AAVrh.32.33 with a 50% decline from peak for all vectors. Stable expression of the minimally immunogenic transgene cFIX from AAVrh.32.33 supports the hypothesis that short-term expression seen with the GFP version of this vector was due to cytotoxic T lymphocytes to this more immunogenic transgene product. Furthermore, for all seven candidates, none of the day 28 and 83 samples tested by the immunocapture assay had detectable cFIX-specific antibodies.

The distribution of vector genomes in organs other than liver (i.e., heart, kidney, lung, pancreas, spleen, brain, muscle, and testis) was assessed by Taqman analysis of total genomic DNA harvested at day 120 (Table 2). The analysis included a rank order of each vector across each tissue with a higher DNA copy number assigned a smaller rank number (i.e., less desirable). Vector abundance in the liver was 100-fold higher than for any other organ for all vectors except AAVrh.32.33 in which the vector was prevalent in equal copy numbers for liver, heart, and lung. Of the remaining six vectors, the one that stood out was AAV6.2 that had significant extrahepatic vector dissemination. Data obtained with AAVrh.32.33 at day 120 may not reflect the initial distribution of vector due to the apparent emergence of destructive cytotoxic T lymphocytes.

Table 2.

Biodistribution of vector DNA in tissues at day 120 following intravenous administration of 1 × 1011 GC of AAV.CB.EGFP

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Discussion

The goal of this study was to identify a cadre of AAV capsids that have the most favorable biological properties as vectors for liver-directed gene transfer. Seven candidates were subjected to rigorous and quantitative analyses measuring the most important properties relevant to efficacy and safety. Each aspect of vector biology noted in Table 1 was given equal weight, and the overall score was determined by the sum of the individual scores with the highest being the most desirable. The overall scores segregated into three groups: best—AAVhu.37 (1.5) and AAVrh.8 (0.5); middle—AAV8 and AAVrh.64R1 (−2) and AAV7 (−4); and worst—AAV6.2 (−12) and AAVrh.32.33 (−17.5). The study was successful in that it clearly stratified the seven candidate vectors over a broad range of cumulative scores. It is worth noting that these candidates were selected from a larger group that had been prescreened for high performance.10

The mouse was used to perform our initial characterization of the seven capsid candidates, realizing that mice may not precisely predict the biology of vectors in primates. We decided that it was best to begin with a model that would permit studies that were capable of measuring multiple parameters with sufficient numbers of animals to yield statistically significant conclusions. Prior data in our laboratory and by others suggest a relatively good correlation between mouse and NHPs when studying some aspects of vector biology.18 We have shown consistent similarities in transduction efficiencies between mice and NHPs when pre-existing NAbs are rigorously excluded. Generation of capsid T cells to AAV2, and not AAVs 7 and 8 was demonstrated both in mice and in NHPs.15,19 Criteria of seroprevalence and inhibition of in vivo transduction are model independent. The one area of difference relates to transgene T-cell responses that seem exaggerated in NHPs as compared to mice when ubiquitous promoters are used. This is why we conducted the T-cell studies with the highly immunogenic transgene product—GFP—from a ubiquitous promoter.

In evaluating the mouse data, several themes emerged. Vectors did not segregate much based on transduction efficiency except for AAVrh.32.33 that showed reduced transduction based on expression of two transgenes, GFP and factor IX. This hierarchy may be influenced by dose because the poorer performance of AAVrh.32.33 was no longer evident at a tenfold lower dose of the GFP vectors. The relatively small differences in transduction efficiency observed across all seven AAV candidates was not surprising because they were selected based in part on pilot expression studies of a larger number of capsid variants. Evaluation of expression at longer time points demonstrated a substantial decline from AAVrh.32.33 when a highly immunogenic transgene such as GFP was used that appears to be due to the appearance of destructive cytotoxic T lymphocytes that is consistent with the use of this vector as a T-cell vaccine.20 Liver pathology did not correlate with LFTs that could reflect difficulties in quantifying histopathology or the presence of nondestructive infiltration of inflammatory cells with some vectors. However, the most severe elevation of LFTs was associated with the most robust activation of transgene specific T cells as observed with AAVrh.32.33. Importantly, capsid-specific T cells, as best demonstrated with AAV6.2, had no effect on stability of transgene expression or LFT abnormalities. The one finding that remains unexplained is the late decline in transgene expression from AAV7.

Our future plan is to use the relative overall ranking provided in Table 1 to further evaluate the three candidates selected herein, AAVhu.37, AAVrh.8, and AAV8, in NHP studies. The NHP studies will be necessary in selecting the capsid among the three finalists that will progress to clinical applications while providing the context for assessing the utility of mouse data in studying vector biology. This kind of information is critical when assessing vector efficacy in relevant animal models of human diseases that are prerequisite for initiating clinical trials and, for the most part, can only be performed in mice.

Materials and Methods

Vector construction, production, and purification. Recombinant AAV vectors expressing enhanced GFP driven by cytomegalovirus-enhanced chicken β-actin promoter (AAV.CB.EGFP) or cFIX driven by liver-specific promoter (AAV.LSP.cFIX-W), and packaged with viral capsids from AAV8, 6.2, 7, rh.64R1, hu.37, rh.8, rh.32.33, or AAV2 used in this study were produced by the Penn Vector Core at the University of Pennsylvania as described previously.21 AAV capsid sequences of natural isolates or carrying corrected singletons were described previously.8,9,10,22 All vectors used in this study were purified by two rounds of cesium chloride gradient centrifugation, buffer-exchanged with phosphate-buffered saline, and concentrated using Amicon Ultra 15 centrifugal filter devices-100K (Millipore, Bedford, MA). Genome titer (genome copy/ml) of AAV vectors was determined by real-time PCR using a primer/probe set corresponding to the polyA region of the vector and linearized plasmid standards. All vectors used in this study passed the endotoxin assay using QCL-1000 chromogenic LAL test kit (Cambrex Bio Science, Walkersville, MD).

Murine experiments. CB6F1 and C57BL/6 male mice (6–8 weeks old) were purchased from Charles River Laboratories (Wilmington, MA) and kept at the Animal Facility of the Translational Research Laboratories at the University of Pennsylvania. All animal procedures were performed in accordance with protocols approved by the Institute of Animal Care and Use Committees at the University of Pennsylvania. For GFP experiments, CB6F1 male mice received a single tail vein injection of 1011 genome copies of vector packaged with each of the seven candidate capsids. Serum transaminase levels were monitored throughout the experiment (Antech Diagnostics, Irvine, CA). At days 7, 14, 28, 90, and 120 following vector administration, a subset of six mice was sacrificed from each group for evaluation of GFP expression in liver, liver histology, and T-cell responses to AAV capsid and GFP in spleen and liver. Vector biodistribution studies were performed on tissues including liver, heart, kidney, lung, pancreas, spleen, brain, muscle, and testis from mice sacrificed at day 120 following vector administration.

For passive transfer experiments, C57BL/6 male mice received two intravenous injections at 24 and 2 hours prior to vector administration, and received a total of 40, 12, 4, 1.2, or 0 mg of human IVIG Carimune NF (CSL Behring, Bern, Switzerland). Two hours after the last IVIG passive transfer, a group of five mice from each IVIG dose group received an intravenous injection of 1 × 1011 genome copies of AAV.LSP.cFIX-W vector packaged with each of the seven candidate capsids, as well as AAV2 for comparison. cFIX expression levels in the plasma following vector administration were determined by enzyme-linked immunosorbent assay (ELISA) as described previously.23 Immunocapture assay for detection of anti-cFIX IgG was performed as published using immunoglobulin standard from Sigma-Aldrich (St Louis, MO).23

Quantification of GFP expression in liver and pathology. To visualize GFP expression in liver, tissues were fixed overnight in formalin, washed in phosphate-buffered saline for 30 min, and frozen in O.C.T compound (Sakura Finetek USA, Torrance, CA). Cryosections were prepared at 8 µm. To quantify GFP expression, representative images from each liver were taken at identical camera and microscope settings with a fluorescence microscope equipped with a digital camera. For every image, the brightness values were measured for all pixels with ImageJ software (Rasband 1997–2006; National Institutes of Health, Bethesda, MD, http://rsb.info.nih.gov/ij/) and then for each brightness value between the background level and the maximum value (255), the number of corresponding pixels was determined. The number of pixels was then multiplied with their brightness value, and the products were added to give a final value for GFP intensity for each liver.

Hematoxylin and eosin–stained sections from formalin-fixed paraffin-embedded liver samples were scored from 0 (normal) to 3 (strongest) for portal and lobular inflammation, respectively. The score was mainly based on the frequency as well as the size of infiltration sites in either portal or lobular liver areas. We only very rarely observed single mitotic or apoptotic hepatocytes, and therefore did not include the presence of those cells into the score.

IFN-γ ELISPOT assay. Lymphocytes (2 × 105) isolated from spleen or liver were stimulated with 2 µg/ml of GFP peptide (LPDNHYLSTQSALSK) and capsid peptide (IPQYGYLTL for AAV8, 6.2, 7, hu.37, and rh.64R1, VPQYGYLTL for AAVrh.8). For AAVrh.32.33 capsid, because the dominant H-2d-restricted epitope was unknown at the time of this study, cells were stimulated with a 50-peptide pool (pool B, 15-mers with a 10-amino-acid overlap with the preceding peptide, covering amino acids 251–510 of VP1; Mimotopes, Clayton, Australia). IFN-γ ELISPOT assay was performed according to manufacturer's instructions (BD Pharmingen, San Diego, CA). Spots were counted with an AID ELISPOT reader system (Autoimmun Diagnostika, Strassberg, Germany). Peptide-specific cells were represented as spot-forming cells per 106 splenocytes and were calculated by subtracting spot numbers in wells containing only medium from spot numbers in peptide-containing wells.

AAV NAb assay. Serum samples were heat inactivated at 56 °C for 30 minutes. NAb assays were performed on Huh7 cells as previously described.6

Vector biodistribution analysis. Tissue DNAs were extracted using QIAamp DNA Mini Kit (Qiagen, Valencia, CA). Detection and quantification of vector genomes in extracted DNA were performed by real-time PCR as described previously.24

Statistical analysis and scoring system. The candidate vectors were rated by using a scoring system that incorporated measurements of each of the following criteria: gene expression, stability of gene expression, host liver function, host liver pathology, host T-cell immune responses to vector and to transgene, prevalence of NAb in humans, and vector biodistribution outside of liver. When goodness of fit tests were used to evaluate several of these measured criteria, the data were found to be not normally distributed, so we opted to use the median +/− robust 95% confidence intervals as the statistic of choice in the following manner.25,26,27,28 For a given criterion, the central tendency for all of the measurements across all vectors was determined. The vector with the narrowest confidence interval whose median score was closest to the central tendency was chosen to be the reference vector. This reference vector was assigned a score of 0. Any vector whose confidence interval overlapped the reference median was also assigned a score of 0 for that criterion. Integer scores ranging from −5 to +5 were assigned based on the distance of the median ± confidence interval for a particular vector from the central tendency. Scores for each criterion were kept in the range from −5 to +5 to prevent any single score from being overly dominant in determining the final score. This was repeated for each criterion, and the final score for each vector was then determined by summing the values given to each vector for the scoring criteria.

Acknowledgments

We thank Julie Johnston and Arbans Sandhu (Penn Vector Core) for supplying vectors; Deirdre McMenamin and Regina Munden for invaluable assistance with animal studies (Gene Therapy Program); and Di Wu (Gene Therapy Program) for tissue sectioning. We thank Jiyang Shen for developing a macro program to allow analysis of green fluorescent protein expression in liver. This work was supported in part by the Kettering Family Foundation and the following grants to J.M.W.: P01-HD057247, P01-HL059407, P30-DK047757, and GlaxoSmithKline. H.W. was supported by a scholarship from China Scholarship Council. L.H.V. is an inventor on patents licensed to various biopharmaceutical companies, including ReGenX. J.M.W. is a consultant to ReGenX Holdings, and is a founder of, holds equity in, and receives a grant from affiliates of ReGenX Holdings; in addition, he is an inventor on patents licensed to various biopharmaceutical companies, including affiliates of ReGenX Holdings.

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