RESUMO
The nature of viral vectors is suggested to be a significant contributor to undesirable immune responses subsequent to gene transfer. Such viral vectors, recognized as danger signals by the host immune system, activate dendritic cells (DCs), causing unwanted antivector and/or transgene product immunity. We recently reported efficient induction of immune tolerance to coagulation factor IX (FIX) by direct intramuscular injection of adeno-associated virus (AAV)-FIX. AAV vectors are nonpathogenic and elicit minimal inflammatory response. We hypothesized that the nonpathogenic nature of AAV plays a critical role in induction of tolerance after AAV gene transfer. We observed inefficient recruitment and activation of DCs subsequent to intramuscular injection of AAV. To further validate our hypothesis, we examined immune responses to FIX after intramuscular injection of AAV with simultaneous activation of DCs. We were able to achieve phenotypic and functional activation of DCs after administration of lipopolysaccharide and anti-CD40 antibody. However, we observed efficient induction of FIX tolerance irrespective of DC activation in mice with different genetic and major histocompatibility complex backgrounds. Furthermore, activation of DCs did not exaggerate the immune response induced after intramuscular injection of AAV serotype 2 vector. Our results demonstrate that induction of FIX tolerance after AAV gene transfer is independent of DC activation status.
Assuntos
Células Dendríticas/imunologia , Fator IX/genética , Vetores Genéticos/administração & dosagem , Tolerância Imunológica/genética , Animais , Células Cultivadas , Células Dendríticas/metabolismo , Dependovirus/genética , Eficiência , Fator IX/administração & dosagem , Técnicas de Transferência de Genes , Injeções Intramusculares , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Fatores de Tempo , Transdução Genética/métodosRESUMO
Conventional gene therapy of hemophilia A relies on the transfer of factor VIII (FVIII; encoded by the F8 gene) cDNA. We carried out spliceosome-mediated RNA trans-splicing (SMaRT) to repair mutant FVIII mRNA. A pre-trans-splicing molecule (PTM) corrected endogenous FVIII mRNA in F8 knockout mice with the hemophilia A phenotype, producing sufficient functional FVIII to correct the hemophilia A phenotype. This is the first description of phenotypic correction of a genetic defect by RNA repair in a knockout animal model. Our results indicate the feasibility of using SMaRT to repair RNA for the treatment of genetic diseases.
Assuntos
Fator VIII/genética , Terapia Genética/métodos , Hemofilia A/genética , Fenótipo , Splicing de RNA , Spliceossomos/metabolismo , Animais , Coagulação Sanguínea/fisiologia , Linhagem Celular , Modelos Animais de Doenças , Fator VIII/metabolismo , Hemofilia A/terapia , Humanos , Camundongos , Camundongos Knockout , RNA/genética , RNA/metabolismoRESUMO
Regulatory T cells play a major role in induction and maintenance of immune tolerance and immunological homeostasis. A variety of strategies have been attempted to induce regulatory T cells for control of unwanted, adverse immunity in autoimmune diseases, transplantation as well as gene transfer. We recently reported efficient induction of immune tolerance to coagulation factor IX (FIX) following intramuscular AAV1 gene transfer. In the current study, we performed a systematic and comprehensive examination of the role and function of regulatory T cells in induction and maintenance of FIX tolerance in the context of intramuscular AAV1 gene transfer. We observed no significant upregulation of regulatory T cells in the FIX-tolerant mice. In addition, adoptive transfer of splenocytes from FIX-tolerant mice did not suppress anti-hFIX immunity in recipient mice. Both in vitro and in vivo depletion of regulatory T cells failed to reverse FIX tolerance. These observations revealed that regulatory T cells do not play a significant role in the maintenance/protection of the established FIX tolerance. Our results provide critical insight into the role and function of regulatory T cells in induction and maintenance/protection of immune tolerance in gene transfer, complementing the current paradigm of immune tolerance mechanism.
Assuntos
Dependovirus/genética , Fator IX/imunologia , Tolerância Imunológica/imunologia , Linfócitos T Reguladores/imunologia , Animais , Proliferação de Células , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Fator IX/genética , Citometria de Fluxo , Vetores Genéticos/genética , Humanos , Tolerância Imunológica/genética , CamundongosRESUMO
Direct intramuscular injection (IM) of adeno-associated virus (AAV) has been proven a safe and potentially efficient procedure for gene therapy of many genetic diseases including hemophilia B. It is, however, contentious whether high antigen level induces tolerance or immunity to coagulation factor IX (FIX) following IM of AAV. We recently reported induction of FIX-specific immune tolerance by IM of AAV serotype one (AAV1) vector in mice. We hypothesize that the expression of high levels of FIX is critical to induction of FIX tolerance. In this study, we investigated the correlation among AAV dose, FIX expression, and tolerance induction. We observed that induction of immune tolerance or immunity to FIX was dependent on the dose of AAV1-human FIX (hFIX) given and the level of FIX antigen expressed in both normal and hemophilia mice. We then defined the minimum AAV1-hFIX dose and the lowest level of FIX needed for FIX tolerance. Different from hepatic AAV-hFIX gene transfer, we found that FIX tolerance induced by IM of AAV1 was not driven by regulatory T cells. These results provided further insight into the mechanism(s) of FIX tolerance, contributing to development of hemophilia gene therapy, and optimization of FIX tolerance induction protocols.
Assuntos
Dependovirus/genética , Fator IX/genética , Fator IX/imunologia , Vetores Genéticos/genética , Tolerância Imunológica/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Citometria de Fluxo , Vetores Genéticos/administração & dosagem , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
The mainstay of gene transfer studies is the use of wild-type cDNAs to effect phenotypic correction of diseases. However, this strategy is not feasible for genetic diseases caused either by mutations of large genes or by dominant-negative mutations, or where the regulation of the gene is critical. In this review, we will discuss a novel RNA reprogramming strategy - spliceosome-mediated RNA trans-splicing - where the pre-messenger RNA is modified by the splicing of two independent RNA species. The use of trans-splicing to effect phenotypic change in the hereditary bleeding disorder haemophilia A will be discussed.
Assuntos
Terapia Genética/métodos , Hemofilia A/terapia , Precursores de RNA/genética , Trans-Splicing , Animais , Humanos , Camundongos , Spliceossomos/metabolismoRESUMO
Hemophilia results from a deficiency of coagulation Factor VIII or IX and manifests clinically as spontaneous bleeding into the large joints and soft tissue. Current treatment relies on the intravenous infusion of recombinant or purified Factor proteins. Factor infusion is effective, but transient due to the short half-life of Factor proteins. Recent developments in gene transfer technology have led to new strategies using molecular therapeutics as permanent treatment for bleeding disorders. This review describes recent novel molecular strategies for the treatment of the hemophilias.
Assuntos
Dependovirus/genética , Terapia Genética , Vetores Genéticos , Hemofilia A/terapia , RNA/genética , Humanos , Splicing de RNARESUMO
Adeno-associated viral (AAV) vector is attracting significant interest for use in gene therapy for genetic diseases, because of its unique and advantageous characteristics, compared to other currently available viral vectors. Eight natural serotypes of AAV have been identified, of which AAV serotype 2 is the one best characterized and most widely used in current gene delivery studies. The application of AAV serotype 2 in hemophilia B gene therapy is a promising development in gene therapy for genetic diseases such as hemophilia. Preliminary studies have demonstrated relation and distinction of host, genome sequences, replication, tropism, packaging of recombinant virions and cross-reactivity of neutralizing antibodies among different serotypes of AAV. This review summarizes the progress of studies in AAV serotypes and pertinent applications in hemophilia B gene therapy. The latest progress in gene delivery of coagulant factor IX (for hemophilia B) using AAV serotype vectors is described in detail.
Assuntos
Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos , Hemofilia B/terapia , Adenoviridae/genética , Animais , Técnicas de Transferência de Genes , Vírus Auxiliares , Hemofilia B/genética , HumanosRESUMO
Adeno-associated virus (AAV) is a useful vector for hemophilia gene therapy, but the limited effective packaging capacity of AAV (5 kb) appears to be incompatible with factor VIII (gene symbol F8) cDNA (7 kb). Although we previously demonstrated efficient packaging and expression of B-domain-deleted human F8 (BDD-F8) using a single AAV vector, the packaging limit still excludes the use of large/strong regulatory elements. Here we exploited the split AAV vector technology that expands the packaging capacity of AAV through head-to-tail dimerization. To test the feasibility of AAV heterodimerization for F8 expression, we generated a 5' vector that includes a large enhancer/promoter cassette linked with exons 1-12 of the F8 cDNA and a half-intron-carrying splice donor site. A complementing 3' vector contains another half-intron-carrying splice acceptor site linked with the remaining F8 cDNA and a polyadenylation signal. Following coinfection of 293 and HepG2 cells, the 5' and 3' vectors together produced functional human factor VIII protein at a level of 120 mU/ml (24 ng/ml). No factor VIII protein was detected if only one of the vectors was used. Correct head-to-tail vector dimerization as well as spliced BDD-F8 mRNA was detected by DNA PCR and RT-PCR, respectively. Furthermore, intraportal injection of two rAAV/F8 vectors in immunodeficient mice produced 2% of the normal level of factor VIII for four months. Our results demonstrate the potential use of AAV dimerization for F8 expression.