RESUMO
Mice carrying mutations in both the dystrophin and utrophin genes die prematurely as a consequence of severe muscular dystrophy. Here, we show that intravascular administration of recombinant adeno-associated viral (rAAV) vectors carrying a microdystrophin gene restores expression of dystrophin in the respiratory, cardiac and limb musculature of these mice, considerably reducing skeletal muscle pathology and extending lifespan. These findings suggest rAAV vector-mediated systemic gene transfer may be useful for treatment of serious neuromuscular disorders such as Duchenne muscular dystrophy.
Assuntos
Dependovirus/genética , Distrofina/genética , Técnicas de Transferência de Genes , Músculo Esquelético/fisiopatologia , Distrofia Muscular Animal/terapia , Músculos Respiratórios/fisiopatologia , Animais , Distrofina/uso terapêutico , Vetores Genéticos , Longevidade , Camundongos , Fibras Musculares Esqueléticas/fisiologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/fisiopatologiaRESUMO
A major obstacle limiting gene therapy for diseases of the heart and skeletal muscles is an inability to deliver genes systemically to muscles of an adult organism. Systemic gene transfer to striated muscles is hampered by the vascular endothelium, which represents a barrier to distribution of vectors via the circulation. Here we show the first evidence of widespread transduction of both cardiac and skeletal muscles in an adult mammal, after a single intravenous administration of recombinant adeno-associated virus pseudotype 6 vectors. The inclusion of vascular endothelium growth factor/vascular permeability factor, to achieve acute permeabilization of the peripheral microvasculature, enhanced tissue transduction at lower vector doses. This technique enabled widespread muscle-specific expression of a functional micro-dystrophin in the skeletal muscles of dystrophin-deficient mdx mice, which model Duchenne muscular dystrophy. We propose that these methods may be applicable for systemic delivery of a wide variety of genes to the striated muscles of adult mammals.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Terapia Genética/métodos , Vetores Genéticos/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/terapia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Cromatografia de Afinidade , Primers do DNA , Modelos Animais de Doenças , Distrofina/metabolismo , Endotélio/metabolismo , Vetores Genéticos/administração & dosagem , Injeções Intravenosas , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Contração Muscular/fisiologia , Parvovirus/metabolismoRESUMO
Hepatitis delta virus (HDV) can dramatically worsen liver disease in patients coinfected with hepatitis B virus (HBV). No effective medical therapy exists for HDV. The HDV envelope requires HBV surface antigen proteins provided by HBV. Once inside a cell, however, HDV can replicate its genome in the absence of any HBV gene products. In vitro, HDV virion assembly is critically dependent on prenyl lipid modification, or prenylation, of its nucleocapsid-like protein large delta antigen. To overcome limitations of current animal models and to test the hypothesis that pharmacologic prenylation inhibition can prevent the production of HDV virions in vivo, we established a convenient mouse-based model of HDV infection capable of yielding viremia. Such mice were then treated with the prenylation inhibitors FTI-277 and FTI-2153. Both agents were highly effective at clearing HDV viremia. As expected, HDV inhibition exhibited duration-of-treatment dependence. These results provide the first preclinical data supporting the in vivo efficacy of prenylation inhibition as a novel antiviral therapy with potential application to HDV and a wide variety of other viruses.
Assuntos
Vírus Delta da Hepatite/efeitos dos fármacos , Metionina/análogos & derivados , Prenilação de Proteína/efeitos dos fármacos , Alquil e Aril Transferases/antagonistas & inibidores , Animais , Antivirais/química , Antivirais/farmacologia , Modelos Animais de Doenças , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Farnesiltranstransferase , Hepatite B/complicações , Antígenos de Superfície da Hepatite B/genética , Hepatite D/complicações , Hepatite D/tratamento farmacológico , Hepatite D/virologia , Vírus Delta da Hepatite/genética , Vírus Delta da Hepatite/fisiologia , Humanos , Metionina/química , Metionina/farmacologia , Camundongos , Camundongos Transgênicos , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Edição de RNA , Viremia/tratamento farmacológico , Replicação Viral/efeitos dos fármacosRESUMO
A major limitation of adenovirus-mediated gene therapy for inherited diseases is the instability of transgene expression in vivo, which originates at least in part from the loss of the linear, extrachromosomal vector genomes. Herein we describe the production of a gene-deleted adenovirus-transposon vector that stably maintains virus-encoded transgenes in vivo through integration into host cell chromosomes. This system utilizes a donor transposon vector that undergoes Flp-mediated recombination and excision of its therapeutic payload in the presence of the Flp and Sleeping Beauty recombinases. Systemic in vivo delivery of this system resulted in efficient generation of transposon circles and stable transposase-mediated integration in mouse liver. Somatic integration was sufficient to maintain therapeutic levels of human coagulation Factor IX for more than six months in mice undergoing extensive liver proliferation. These vectors combine the versatility of adenoviral vectors with the integration capabilities of a eukaryotic DNA transposon and should prove useful in the treatment of genetic diseases.
Assuntos
Adenoviridae/genética , Elementos de DNA Transponíveis/genética , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Fígado/enzimologia , Transgenes/genética , Animais , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Transposases/classificação , Transposases/genéticaRESUMO
We used the integrase from phage phiC31 to integrate the human Factor IX (hFIX) gene permanently into specific sites in the mouse genome. A plasmid containing attB and an expression cassette for hFIX was delivered to the livers of mice by using high-pressure tail vein injection. When an integrase expression plasmid was co-injected, hFIX serum levels increased more than tenfold to approximately 4 microg/ml, similar to normal FIX levels, and remained stable throughout the more than eight months of the experiment. hFIX levels persisted after partial hepatectomy, suggesting genomic integration of the vector. Site-specific integration was proven by characterizing and quantifying genomic integration in the liver at the DNA level. Integration was documented at two pseudo-attP sites, native sequences with partial identity to attP, with one site highly predominant. This study demonstrates in vivo gene transfer in an animal by site-specific genomic integration.
Assuntos
Fator IX/biossíntese , Fator IX/genética , Fígado/metabolismo , Mutagênese Sítio-Dirigida , Animais , Regulação da Expressão Gênica , Terapia Genética/métodos , Genoma , Injeções Intravenosas , Integrases/genética , Integrases/metabolismo , Fígado/efeitos dos fármacos , Fígado/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos/administração & dosagem , Plasmídeos/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
While naked DNA gene transfer in vivo usually results in transient gene expression, in some cases long-term transgene expression can be achieved. Here we demonstrate that cis-acting DNA elements flanking the transgene expression cassette and components in the plasmid backbone can significantly influence expression levels from nonviral vectors. To demonstrate this, we administered our most robust human coagulation factor IX (hFIX) expression cassette placed in two different plasmid backbones, into the livers of mice, by hydrodynamic transfection. We found that placing the expression cassette within a minimal plasmid vector pHM5, a modified version of pUC19, resulted in 10 times higher serum hFIX expression levels (up to 20000 ng/ml, 400% of normal hFIX serum levels), compared to a pBluescript backbone. To optimally increase expression levels from a nonviral vector, we added matrix attachment regions (MARs) as cis-acting DNA elements flanking the hFIX expression cassette. We detected five fold higher hFIX expression levels in vivo for up to 1-year posttransfection from a vector that contained the chicken MAR from the lysozyme locus. Together, the present work demonstrates that in addition to the transgene expression cassette, cis-acting DNA elements within and outside of the plasmid backbone need to be evaluated to achieve optimal expression levels in a nonviral gene therapy approach.
Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos , Animais , Fator IX/genética , Hepatócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos/metabolismoRESUMO
We demonstrate that vectors incorporating components from Epstein-Barr virus (EBV) for retention and from human genomic DNA for replication greatly enhance the level and duration of marker gene expression in dividing cultured cells. The same types of vectors were tested in vivo by high-pressure tail vein injection of naked DNA in mice, resulting in liver delivery and expression. The therapeutic gene was a human factor IX (hFIX) minigene comprising genomically derived 5', 3', and intronic sequences that provided relatively good gene expression in vivo. We demonstrated that addition of the EBV EBNA1 gene and its family of repeats binding sites provided a 10- to 100-fold increase in prolonged hFIX expression in mouse liver. A single 25-microg dose of vector DNA generated normal (>5 microg/mL) levels of hFIX throughout the 8 month duration of the experiment. Vector DNA with or without the EBV sequences was retained in liver cells, and vector replication was not a factor in these nondividing liver cells. Instead, it appears that enhancement of stable hFIX expression by the EBV components was responsible for the increased level and duration of therapeutic gene expression. The EBV sequences also significantly enhanced stable expression of a vector carrying the full genomic hFIX gene delivered to mouse liver. These results underline the crucial importance of appropriate gene expression signals on gene therapy vectors and the utility of EBV sequences in particular for increasing stable gene expression.
Assuntos
Fator IX/biossíntese , Regulação da Expressão Gênica/genética , Herpesvirus Humano 4/genética , Rim/metabolismo , Transfecção/métodos , Animais , Clonagem Molecular , DNA Viral/administração & dosagem , Fator IX/análise , Fator IX/genética , Estudos de Viabilidade , Terapia Genética/métodos , Vetores Genéticos , Hemofilia B/tratamento farmacológico , Hepatócitos/metabolismo , Herpesvirus Humano 4/metabolismo , Humanos , Rim/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Controle de Qualidade , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
Systemic delivery of recombinant adeno-associated virus (rAAV) 6 vectors mediates efficient transduction of the entire striated musculature, making this an attractive strategy for muscle gene therapy. However, owing to widespread transduction of non-muscle tissues, optimization of this method would benefit from the use of muscle-specific promoters. Most such promoters either lack high-level expression in certain muscle types or are too large for inclusion in rAAV vectors encoding microdystrophin. Here, we describe novel regulatory cassettes based on enhancer/promoter regions of murine muscle creatine kinase (CK) and alpha-myosin heavy-chain genes. The strongest cassette, MHCK7 (770 bp), directs high-level expression comparable to cytomegalovirus and Rous sarcoma virus promoters in fast and slow skeletal and cardiac muscle, and low expression in the liver, lung, and spleen following systemic rAAV6 delivery in mice. Compared with CK6, our previous best cassette, MHCK7 activity is approximately 400-, approximately 50-, and approximately 10-fold higher in cardiac, diaphragm, and soleus muscles, respectively. MHCK7 also directs strong microdystrophin expression in mdx muscles. While further study of immune responses to MHCK7-regulated microdystrophin expression is needed, this cassette is not active in dendritic cell lines. MHCK7 is thus a highly improved regulatory cassette for experimental studies of rAAV-mediated transduction of striated muscle.
Assuntos
Dependovirus/genética , Vetores Genéticos/genética , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Clonagem Molecular/métodos , Creatina Quinase/genética , Creatina Quinase/metabolismo , Imunofluorescência , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/citologia , Miocárdio/citologia , Regiões Promotoras Genéticas/genética , Transfecção , Miosinas Ventriculares/genética , Miosinas Ventriculares/metabolismoRESUMO
A promising approach for treating Duchenne muscular dystrophy (DMD) is by autologous cell transplantation of myogenic stem cells transduced with a therapeutic expression cassette. Development of this method has been hampered by a low frequency of cellular engraftment, the difficulty of tracing transplanted cells, the rapid loss of autologous cells carrying marker genes that are unable to halt muscle necrosis and the difficulty of stable transfer of a large dystrophin gene into myogenic stem cells. We engineered a 5.7 kb miniDys-GFP fusion gene by replacing the dystrophin C-terminal domain (DeltaCT) with an eGFP coding sequence and removing much of the dystrophin central rod domain (DeltaH2-R19). In a transgenic mdx(4Cv) mouse expressing the miniDys-GFP fusion protein under the control of a skeletal muscle-specific promoter, the green fusion protein localized on the sarcolemma, where it assembled the dystrophin-glycoprotein complex and completely prevented the development of dystrophy in transgenic mdx(4Cv) muscles. When myogenic and other stem cells from these mice were transplanted into mdx(4Cv) recipients, donor cells can be readily identified in skeletal muscle by direct green fluorescence or by using antibodies against GFP or dystrophin. In mdx(4Cv) mice reconstituted with bone marrow cells from the transgenic mice, we monitored engraftment in various muscle groups and found the number of miniDys-GFP(+) fibers increased with time. We suggest that these transgenic mdx(4Cv) mice are highly useful for developing autologous cell therapies for DMD.
Assuntos
Distrofina/genética , Terapia Genética , Proteínas de Fluorescência Verde/genética , Distrofia Muscular de Duchenne/genética , Proteínas Recombinantes de Fusão/genética , Animais , Distrofina/biossíntese , Camundongos , Camundongos Transgênicos , Contração Muscular , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/terapia , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Sarcolema/metabolismo , Transplante de Células-TroncoRESUMO
Hepatitis C virus (HCV) is an RNA virus infecting one in every 40 people worldwide. Current treatments are ineffective and HCV is the leading cause of liver failure leading to transplantation in the United States and Europe. Translational control of HCV is a prime therapeutic target. We assessed the inhibitory potential of morpholino phosphoramidate antisense oligonucleotides (morpholinos) on HCV translation by codelivering them with reporter plasmids expressing firefly luciferase under the translational control of the HCV internal ribosome entry site (IRES) into the livers of mice. Real-time imaging of HCV IRES luciferase reporter messenger RNA (mRNA) translation in living mice showed that a 20-mer complementary to nucleotides 345-365 of the IRES inhibited translation by greater than 95% for at least 6 days and showed mismatch specificity. No significant nonspecific inhibition of a cap-dependent luciferase or encephalomyocarditis virus (EMCV) IRES luciferase reporter translation was observed. Inhibition by the 20-mer morpholino was dose dependent, with 1 nmol/mouse giving the highest inhibition. In conclusion, morpholino antisense oligonucleotides are potent inhibitors of HCV IRES translation in a preclinical mouse model; morpholinos have potential as molecular therapeutics for treating HCV and other viral infections. The in vivo model described is a broadly applicable, straightforward, and rapid readout for inhibitor efficacy. As such, it will greatly facilitate the development of novel therapeutic strategies for viral hepatitis. Notably, the level of antisense inhibition observed in this in vivo model is similar to the maximal inhibition we have obtained previously with RNA interference in mice.
Assuntos
Hepacivirus/genética , Hepatite C/terapia , Oligonucleotídeos Antissenso/farmacologia , Biossíntese de Proteínas/genética , Animais , Feminino , Terapia Genética , Hepacivirus/crescimento & desenvolvimento , Luciferases/genética , Camundongos , Plasmídeos , Ribossomos/genética , TransfecçãoRESUMO
Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in vivo. In hepatocyte nuclei, the incoming single-stranded (ss) vector genomes are converted into various forms of double-stranded (ds) genomes including extrachromosomal linear and circular monomers and concatemers, and a small portion of the vector genomes integrate into chromosomes. The mechanism of genome conversion is not well understood. In the present study, we analyzed the role of inverted terminal repeat (ITR) sequences of ds circular or linear rAAV vector intermediates in concatemerization. We synthesized supercoiled ds circular monomers with a double-D ITR (DDITR) (C+), and ds linear monomers with an ITR at each end (L+), and their control molecules, C- and L-, which lack the ITR-derived sequences, and transfected mouse hepatocytes with these molecules in vivo to assess their capacity for concatemerization. The transfected L+ or L-, but not C+ or C- molecules, concatemerized in vivo irrespective of the presence or absence of the ITRs. In addition, our results suggested that transfected C+ or C- species were not efficient substrates for integration. Based on these observations, we propose a model whereby ds linear molecules with free DNA ends, but not circular molecules, play an important role in rAAV vector genome concatemerization.
Assuntos
DNA/química , Dependovirus/genética , Vetores Genéticos , Animais , Fator IX/administração & dosagem , Fator IX/genética , Feminino , Genoma , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
RNA interference is an evolutionarily conserved surveillance mechanism that responds to double-stranded RNA by sequence-specific silencing of homologous genes. Here we show that transgene expression can be suppressed in adult mice by synthetic small interfering RNAs and by small-hairpin RNAs transcribed in vivo from DNA templates. We also show the therapeutic potential of this technique by demonstrating effective targeting of a sequence from hepatitis C virus by RNA interference in vivo.
Assuntos
Envelhecimento/genética , Inativação Gênica , Hepacivirus/genética , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Animais , Genes Reporter/genética , Humanos , Fígado/metabolismo , Luciferases/biossíntese , Luciferases/genética , Camundongos , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno , RNA não Traduzido/química , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Especificidade por Substrato , Transgenes/genética , Proteínas não Estruturais Virais/biossíntese , Proteínas não Estruturais Virais/genéticaRESUMO
Transposon-based vectors represent promising new tools for chromosomal transgene insertion and establishment of persistent gene expression in vivo. Here, we report the development of helper-independent transposon-transposase (HITT) vectors, which contain on single plasmids (i) a Sleeping Beauty (SB) transposon containing the transgene and (ii) a SB transposase expression cassette. To obtain an optimal level of transposase expression from HITT vectors, we determined the relative strength of a panel of different promoters in mouse liver and used these promoters to drive transposase expression from injected HITT vectors carrying a human alpha(1)-antitrypsin (hAAT) expression cassette flanked by transposon inverted repeats. By correlating promoter strength with stabilized serum hAAT levels, a narrow expression window supporting high-level transposition in the liver was defined. Peak levels of long-term gene expression were obtained with promoters 30- to 40-fold less active than CMV in mouse liver, whereas reduced stable levels of hAAT were detected with both weaker and stronger promoters. Injected HITT vectors induced transposase-dependent insertion of transposon DNA into the genome of at least 5-6% of transfected hepatocytes, generating levels of persistent hAAT expression that were 2- to 4-fold higher than with an optimized two-plasmid approach. In addition, we show that HITT vectors carrying a human factor IX (hFIX)-containing transposon support (i) long-term hFIX expression in normal mice and (ii) partial phenotypic correction in a mouse model of hemophilia B. SB-based HITT vectors represent a major advance in the establishment of persistent transgene expression from nonviral gene delivery systems and should prove useful for gene transfer to tissues or cell types in which transfection efficiencies are low.
Assuntos
Elementos de DNA Transponíveis , Vetores Genéticos , Plasmídeos , Transposases/genética , Animais , Fator IX/genética , Fator IX/metabolismo , Hemofilia B/tratamento farmacológico , Fígado/metabolismo , Camundongos , Plasmídeos/farmacologia , Regiões Promotoras Genéticas , Transgenes , Transposases/metabolismoRESUMO
Nonviral plasmid DNA is a promising vector for achieving ex vivo and in vivo gene transfer. However, transgene expression is usually transient, especially in dividing target cells due to loss of vector genomes. Here we describe the use of naked double-stranded (ds) linear DNA as a way to insert exogenous DNA sequences into chromosomes of mouse hepatocytes in vivo, without helper components such as integrase or transposase. We constructed ds linear DNA vectors with or without adeno-associated virus inverted terminal repeats (AAV-ITRs), introduced them into mouse hepatocytes in vivo using a hydrodynamics-based transfection technique, and analyzed for vector genome integration in various ways. Surprisingly, these linear DNA molecules integrated in mouse hepatocytes in vivo at a level of 0.3-0.5 vector genome, or more, per diploid genomic equivalent irrespective of the AAV-ITR sequences. Our results establish a novel and simple way to engineer chromosomes in vivo and provide further insights into the mechanisms of recombinant AAV vector integration in vivo. In addition, they may provide a clue for developing new nonviral integrating gene delivery vector systems.
Assuntos
Cromossomos , DNA/administração & dosagem , Dependovirus/genética , Animais , Sequência de Bases , Southern Blotting , DNA/genética , Hepatócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência MolecularRESUMO
Hepatitis C virus (HCV) is an RNA virus infecting 1 in every 40 people worldwide. Development of new therapeutics for treating HCV has been hampered by the lack of small-animal models. We have adapted existing hydrodynamic transfection methods to optimize the delivery of RNAs to the cytoplasm of mouse liver cells in vivo. Transfected HCV genomic RNA failed to replicate in mouse liver, suggesting a post-entry block to viral replication. Real-time imaging of HCV internal ribosome entry site (IRES) firefly luciferase reporter mRNA translation in living mice demonstrated that the HCV IRES was functional in mouse liver. We then used this system as a model for studying HCV RNA translation in mice. We compared translation by several mutant HCV IRES variants in cell lysates, cultured cells, and mouse liver. We measured the contribution to translation of a cap, HCV 3'-untranslated region (UTR), poly(A) tail, domains II, IIIb, IIIabc, IIIabcd, IIId, and the initiator codon. Efficient translation required a 3'-UTR in mice and HeLa cells, but not in rabbit reticulocyte lysates. Translational regulation of transfected RNAs was stringent in mice. The method we describe could be useful for studies in mice of antisense or ribozyme inhibitors targeting the IRES as well as other RNA biochemical studies in vivo.
Assuntos
Modelos Animais de Doenças , Hepacivirus/genética , Biossíntese de Proteínas , Regiões 3' não Traduzidas , Adenosina/genética , Animais , Células HeLa , Hepatite C/virologia , Humanos , Técnicas In Vitro , Fígado/ultraestrutura , Fígado/virologia , Luciferases/metabolismo , Camundongos , Hormônios Placentários/metabolismo , Polímeros , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/metabolismo , RNA Viral/biossínteseRESUMO
We report the generation and use of pseudotyped adeno-associated viral (AAV) vectors for the liver-specific expression of human blood coagulation factor IX (hFIX). Therefore, an AAV-2 genome encoding the hfIX gene was cross-packaged into capsids of AAV types 1 to 6 using efficient, large-scale technology for particle production and purification. In immunocompetent mice, the resultant vector particles expressed high hFIX levels ranging from 36% (AAV-4) to more than 2000% of normal (AAV-1, -2, and -6), which would exceed curative levels in patients with hemophilia. Expression was dose- and time-dependent, with AAV-6 directing the fastest and strongest onset of hFIX expression at all doses. Interestingly, systemic administration of 2 x 1012 vector particles of AAV-1, -4, or -6 resulted in hFIX levels similar to those achieved by portal vein delivery. For all other serotypes and particle doses, hepatic vector administration yielded up to 84-fold more hFIX protein than tail vein delivery, corroborated by similarly increased vector DNA copy numbers in the liver, and elicited a reduced immune response against the viral capsids. Finally, neutralization assays showed variable immunologic cross-reactions between most of the AAV serotypes. Our technology and findings should facilitate the development of AAV pseudotype-based gene therapies for hemophilia B and other liver-related diseases.