Cholesterol metabolism in normal and heterozygous familial hypercholesterolemic newborns

The implantation of nonautologous cells encapsulated in immunoprotective microcapsules provides an alternative nonviral method for gene therapy. This strategy was successful in reversing the disease phenotypes of dwarfism and a lysosomal storage disease, mucopolysaccharidosis VII, in murine models. In this article we implanted transgenic hemophilic B mice with microcapsules enclosing factor IX-secreting C2C12 myoblasts to study the clinical potential of this approach in the treatment of hemophilia. Treated mice showed increased plasma factor IX levels as high as 28 ng of human factor IX per milliliter of plasma and decreased activated thromboplastin times (reduced by 20% to 29%). However, the level of factor IX decreased to baseline levels by day 7, coinciding with emergence of anti-human factor IX antibody, the titer of which increased greater than 10-fold by day 28. Monoclonal anti-CD4 antibodies were used to deplete CD4+ T cells to suppress the immune response against the recombinant factor IX. In the treated hemophilic mice, the anti-factor IX antibody response was totally suppressed to beyond day 28 accompanied by a significant decrease in activated thromboplastin time compared with that seen in untreated hemophilic mice. When the microcapsules were recovered from the intraperitoneal cavity after 38 days of implantation, the encapsulated cells continued to secrete factor IX at preimplantation levels, but both cell viability and microcapsule mechanical stability were reduced. Hence although the polymer chemistry of the microcapsules and cell viability may need to be improved for long-term delivery, nonautologous gene therapy with microencapsulated cells has been shown to be effective, at least for the short-term, in alleviating the hemophilic hemostatic anomaly. Coadministration of an immunosuppressant is effective in inhibiting antibody development against the delivered factor IX and should be considered for recipients at risk of inhibitor development.     

Sustained phenotypic correction of hemophilia a mice following oncoretroviral-mediated expression of a bioengineered human factor VIII gene in long-term hematopoietic repopulating cells.

Hematopoietic stem cells (HSCs) are an attractive target cell population for hemophilia A gene therapy because of their capacity to regenerate the hematolymphoid system permanently following transplantation. Here we transplanted bone marrow (BM) cells transduced with a splicing-optimized MSCV oncoretroviral vector expressing a secretion-improved human factor VIII gene into immunocompromised hemophilic mice that had received a reduced dose conditioning regimen. An enhanced green fluorescent protein (EGFP) reporter gene linked to an encephalomyocarditis virus internal ribosome entry site was incorporated into the vector to allow preselection of transduced cells and facile evaluation of engraftment. Sustained expression of EGFP was demonstrated in the peripheral blood, and therapeutic levels of factor VIII were detected in the plasma of the majority of the recipients for the duration of the observation period (up to 22 weeks). Coordinate expression of factor VIII and EGFP (up to 19 weeks) was transferred to secondary BM transplant recipients, indicating that long-term repopulating HSCs had been successfully gene modified. Notably, the hemophilic phenotype of all treated mice was corrected, thus demonstrating the potential of HSC-directed oncoretroviral-mediated factor VIII gene transfer as a curative therapeutic strategy for hemophilia A.     

Tolerance induction by viral in vivo gene transfer

Treatment of genetic disease by protein or gene replacement therapy is hampered by immune responses to the therapeutic protein. An excellent example is formation of inhibitory antibodies to coagulation factors in treatment of the X-linked bleeding disorder hemophilia. Experiments in murine and canine models of hemophilia B (deficiency in factor IX) have demonstrated sustained therapeutic levels of factor IX transgene expression following hepatic adeno-associated viral gene transfer in animals with deletion and nonsense mutations in the factor IX gene. This article reviews experimental evidence for induction of immune tolerance to the factor IX transgene product by hepatic adeno-associated viral gene transfer, which has been shown to limit T helper cell responses and to substantially reduce the risk of antibody responses. Tolerance induction is associated with activation of regulatory CD4(+) T cells capable of suppressing antibody formation to factor IX protein. Hepatic administration of adeno-associated viral vector expressing ovalbumin in mice transgenic for a T cell receptor specific for this antigen provided direct evidence for induction of CD4(+) T cell tolerance, including T cell anergy and clonal deletion. Taken together, these data indicate the potential for viral in vivo gene transfer not only to provide sustained systemic expression, but moreover to induce immunological hypo-responsiveness to the therapeutic gene product.     

Gene transfer for hemophilia: can therapeutic efficacy in large animals be safely translated to patients?

Gene transfer is a novel area of therapeutics in which the active agent is a nucleic acid rather than a protein or small molecule. As early as 1997, investigators reported long-term expression of therapeutic levels of factor IX using gene transfer techniques in hemophilia B mice, and similar data were thereafter reported in mice with hemophilia A. Efforts to translate these results to hemophilic dog models at first yielded only marginally therapeutic levels (1%-2% normal circulating levels), but within the past few years have achieved levels in the range of 10%-20% through multiple different gene transfer strategies. Early phase clinical testing has revealed that many aspects of gene transfer in humans were accurately predicted by studies in hemophilic dogs, but that other aspects were not, and were only appreciated as a result of clinical testing. Studies in the next few years will determine whether the problems identified in preclinical and early phase clinical testing can be solved to develop a therapeutic gene transfer approach to hemophilia.     

Induction of immune tolerance to coagulation factor IX antigen by in vivo hepatic gene transfer

Gene replacement therapy is an attractive approach for treatment of genetic disease, but may be complicated by the risk of a neutralizing immune response to the therapeutic gene product. There are examples of humoral and cellular immune responses against the transgene product as well as absence of such responses, depending on vector design and the underlying mutation in the dysfunctional gene. It has been unclear, however, whether transgene expression can induce tolerance to the therapeutic antigen. Here, we demonstrate induction of immune tolerance to a secreted human coagulation factor IX (hF.IX) antigen by adeno-associated viral gene transfer to the liver. Tolerized mice showed absence of anti-hF.IX and substantially reduced in vitro T cell responses after immunization with hF.IX in adjuvant. Tolerance induction was antigen specific, affected a broad range of Th cell subsets, and was favored by higher levels of transgene expression as determined by promoter strength, vector dose, and mouse strain. Hepatocyte-derived hF.IX expression induced regulatory CD4(+) T cells that can suppress anti-hF.IX formation after adoptive transfer. With a strain-dependent rate of success, tolerance to murine F.IX was induced in mice with a large F.IX gene deletion, supporting the relevance of these data for treatment of hemophilia B and other genetic diseases.     

Gene therapy progress and prospects: fetal gene therapy--first proofs of concept--some adverse effects

Somatic gene delivery in utero is a novel approach to gene therapy for genetic disease based on the hypothesis that prenatal intervention may avoid the development of severe manifestations of early-onset disease, allow targeting of otherwise inaccessible tissues including expanding stem cell populations, induce tolerance against the therapeutic transgenic protein and thereby provide permanent somatic gene correction. This approach is particularly relevant in relation to prenatal screening programmes for severe genetic diseases as it could offer prevention as a third option to families faced with the prenatal diagnosis of a genetically affected child. Most investigations towards in utero gene therapy have been performed on mice and sheep fetuses as model animals for human disease and for the application of clinically relevant intervention techniques such as vector delivery by minimally invasive ultrasound guidance. Other animals such as dogs may serve as particular disease models and primates have to be considered in immediate preparation for clinical trials. Proof of principle for the hypothesis of fetal gene therapy has been provided during the last 2 years in mouse models for Crigler Najjar Disease, Leber's congenital amaurosis, Pompe's disease and haemophilia B showing long-term postnatal therapeutic effects and tolerance of the transgenic protein after in utero gene delivery. However, recently we have also observed a high incidence of liver tumours after in utero application of an early form of third-generation equine infectious anaemia virus vectors with SIN configuration. These findings highlight the need for more investigations into the safety and the ethical aspects of in utero gene therapy as well as for science-based public information on risks and benefits of this preventive gene therapy approach before application in humans can be contemplated.     

Intra‐articular injection of mesenchymal stem cells expressing coagulation factor ameliorates hemophilic arthropathy in factor VIII‐deficient mice

Summary. Background: Transplantation of cells overexpressing a target protein represents a viable gene therapeutic approach for treating hemophilia. Here, we focused on the use of autologous mesenchymal stem cells (MSCs) expressing coagulation factor for the treatment of coagulation factor VIII (FVIII) deficiency in mice. Methods and Results: Analysis of luciferase gene constructs driven by different promoters revealed that the plasminogen activator inhibitor‐1 (PAI‐1) gene promoter coupled with the cytomegalovirus promoter enhancer region was one of the most effective promoters for producing the target protein. MSCs transduced with the simian immunodeficiency virus (SIV) vector containing the FVIII gene driven by the PAI‐1 promoter expressed FVIII for several months, and this expression was maintained after multiple mesenchymal lineage differentiation. Although intravenous injection of cell supernatant derived from MSCs transduced with an SIV vector containing the FVIII gene driven by the PAI‐1 promoter significantly increased plasma FVIII levels, subcutaneous implantation of the MSCs resulted in a transient and weak increase in plasma FVIII levels in FVIII‐deficient mice. Interestingly, intra‐articular injection of the transduced MSCs significantly ameliorated the hemarthrosis and hemophilic arthropathy induced by knee joint needle puncture in FVIII‐deficient mice. The therapeutic effects of a single intra‐articular injection of transduced MSCs to inhibit joint bleeding persisted for at least 8 weeks after administration. Conclusions: MSCs provide a promising autologous cell source for the production of coagulation factor. Intra‐articular injection of MSCs expressing coagulation factor may offer an attractive treatment approach for hemophilic arthropathy.     

Angiogenesis enhances factor IX delivery and persistence from retrievable human bioengineered muscle implants.

Human muscle progenitor cells transduced with lentiviral vectors secreted high levels of blood clotting factor IX (FIX). When bioengineered into postmitotic myofibers as human bioartificial muscles (HBAMs) and subcutaneously implanted into immunodeficient mice, they secreted FIX into the circulation for >3 months. The HBAM-derived FIX was biologically active, consistent with the cells' ability to conduct the necessary posttranslational modifications. These bioengineered muscle implants are retrievable, an inherent safety feature that distinguishes this "reversible" gene therapy approach from most other gene therapy strategies. When myofibers were bioengineered from human myoblasts expressing FIX and vascular endothelial growth factor, circulating FIX levels were increased and maintained long term within the therapeutic range, consistent with the generation of a vascular network around the HBAM. The present study implicates an important role for angiogenesis in the efficient delivery of therapeutic proteins using tissue engineered stem cell-based gene therapies.     

Efficient induction of immune tolerance to coagulation factor IX following direct intramuscular gene transfer

BACKGROUND: The formation of inhibitory anti-factor IX (anti-FIX) antibodies is a major complication of FIX protein replacement-based treatment for hemophilia B. It is difficult to treat patients with anti-FIX antibodies. Gene therapy is emerging as a potentially effective treatment for hemophilia. Direct i.m. injection of adeno-associated virus (AAV) is a safe and efficient procedure for hemophilia B gene therapy. However, the development of anti-FIX antibodies following i.m. of AAV may impede its application to patients. OBJECTIVE: We aimed to investigate induction of immune tolerance to human FIX (hFIX) by i.m. of AAV1, further validating i.m. of AAV1 for hemophilia B gene therapy. METHODS AND RESULTS: Cohorts of hemostatically normal and hemophilia B mice with diverse genetic and MHC backgrounds received i.m. of AAV-hFIX. Human FIX antigen and anti-hFIX antibodies were examined. I.m. of 1 x 10(11) vector genomes (VG) of AAV2 elicits formation of anti-hFIX antibodies comparable to those by hFIX protein replacement. I.m. of 1 x 10(11) VG of AAV1 results in expression of therapeutic levels of hFIX (up to 950 ng mL(-1), mean = 772 ng mL(-1), SEM +/- 35.7) and hFIX-specific immune tolerance in C57BL/6 mice. CONCLUSIONS: A single i.m. of AAV1 can result in efficient expression of therapeutic levels of hFIX and induction of hFIX tolerance in hemostatically normal and hemophilic B mice. Our results substantiate the prospect of i.m. of AAV1 for hemophilia B gene therapy and FIX tolerance induction.     

Intravenous administration of an E1/E3-deleted adenoviral vector induces tolerance to factor IX in C57BL/6 mice

Inbred immunocompetent C57BL/6 mice have been a favored strain to study transgene expression of human blood coagulation factor IX (hF.IX) from viral vectors because systemic expression of the secreted protein is not limited by antibody responses following intravenous (i.v.) injection of vector. For example, i.v. injection of an adenoviral (Ad) vector results in sustained expression of hF.IX in normal or hemophilic C57BL/6 mice, while anti-hF.IX antibodies rapidly emerge in other strains (Gene Therapy 4: 473; Blood 91: 784). To investigate these observations further, we injected naive C57BL/6 mice and C57BL/6 mice with pre-existing anti-hF.IX with Ad-hF.IX vector via peripheral vein. All mice expressed hF.IX antigen without detectable anti-hF.IX, even when challenged with hF.IX in different immunogenic settings at later time points. Moreover, in mice with pre-existing immunity, anti-hF.IX titers diminished to undetectable levels after i.v. administration of Ad-hF.IX. Lymphocytes from mice that had received Ad-hF.IX i.v. failed to proliferate when stimulated with hF.IX in vitro after the animals had been repeatedly challenged with hF.IX protein formulated in complete Freund's adjuvant. Thus, absence of anti-hF.IX in C57BL/6 mice after i.v. injection of Ad vector is not due to ignorance to the foreign transgene product. Similar experiments in other strains showed that immune tolerance to hF.IX does not correlate with the strain haplotype or expression of IL-10 cytokine. Given the well-documented immunogenicity of the first-generation adenoviral vector, data from C57BL/6 mice may therefore grossly underestimate immunological consequences in certain gene therapy protocols.     

AAV-mediated gene transfer for the treatment of hemophilia B: problems and prospects

Adeno-associated viral vector-mediated gene transfer of coagulation factor IX to the skeletal muscle or to liver has resulted in sustained correction of hemophilia B in mice and dogs. The two initial phase I/II AAV clinical trials for hemophilia B, delivering a factor IX cDNA to skeletal muscle or liver, showed no serious adverse events. Although the muscle trial failed to achieve a therapeutic level of factor IX in the circulation, long-term expression of clotting factor was demonstrated on muscle biopsies taken up to 3 years after vector injection. Administration of vector to liver via the hepatic artery identified a therapeutic dose, which agreed closely with the doses predicted by studies in hemophilic dogs. However, expression in human subjects lasted for only a period of weeks, followed by a gradual decline in factor IX levels accompanied by a self-limited, asymptomatic rise and fall in liver enzymes. Immune responses to vector capsid may account for this difference in outcome between humans and other species. Here we review the results from both preclinical and clinical studies of adeno-associated viral vector gene transfer for hemophilia B, and the problems that have been identified and that must be overcome to achieve successful transduction and sustained expression.     

Long-term transgene expression from plasmid DNA gene therapy vectors is negatively affected by CpG dinucleotides.

CpG-reduced, CMV-based plasmid DNA constructs encoding human alpha-galactosidase A and factor IX were injected into C57Bl/6, BALB/c, and CD1 mice using hydrodynamics-based delivery of plasmid DNA (pDNA), and gene expression was monitored for 6 months. Linearized and supercoiled pDNAs were compared for their abilities to support long-term expression and to generate immune responses to the transgene product. In all mouse strains supercoiled CpG-reduced pDNA encoding alpha-galactosidase A and factor IX generated higher and more sustained levels of circulating gene product than their supercoiled CpG-replete analogs. Linearizing supercoiled CpG-reduced pDNA did not significantly increase levels of circulating gene product beyond levels supercoiled CpG-reduced pDNA could achieve. Linearizing supercoiled CpG-replete pDNA vectors significantly increased expression compared to their supercoiled CpG-replete analogs, but the increase was short-lived or subtherapeutic. Regardless of vector, liver depot expression did not elicit significant antibody responses to human alpha-galactosidase A or factor IX. Taken together, these data suggest that a clinically acceptable hydrodynamics-based approach targeting the liver combined with CpG-reduced pDNA vectors may represent a viable option for individuals with hemophilia, a lysosomal storage disease, or other disease in which prolonged depot expression of a therapeutic protein from the liver is desirable.     

Transient siRNA-mediated attenuation of liver expression from an alpha-galactosidase A plasmid reduces subsequent humoral immune responses to the transgene product in mice.

Hepatocytes are an effective depot for protein production from gene therapy vectors. However, when gene transfer vectors or their delivery induces hepatic inflammation, adaptive immune responses against the transgene product can ensue. In BALB/c mice, hydrodynamic delivery of a CMV-driven plasmid DNA (pDNA) bearing human alpha-galactosidase A (alphagal) to the liver generated antibodies against alphagal. This humoral immune response was more robust in a transgenic knockout for alphagal, the Fabry mouse. The antibody response could be attenuated in both mouse strains by using a promoter more restricted to hepatocytes. In an attempt to reduce further the humoral responses to alphagal, expression from the transgene was attenuated by using siRNA during the period of initial delivery-associated liver inflammation. In both mouse models and with both promoters, codelivering an alphagal siRNA resulted in a 2 log decrease in initial expression that then increased over the next few weeks to levels generated by the pDNA alone. This strategy led to both attenuated antibodies and an immune status approximating "tolerance" to alphagal. Importantly, in the Fabry mouse, an alphagal siRNA together with a hepatocyte-restricted promoter gave minimal anti-alphagal antibodies and profound tolerance, suggesting that such an approach might have clinical utility for genetic diseases.     

Identification of T-cell epitopes in clotting factor IX and lack of tolerance in inbred mice

Summary.  Immune responses to the factor IX protein pose problems for hemophilia B patients who develop antibodies against factor IX and for potential future treatment with gene therapy. To better define the response to human factor IX, we analyzed T-cell responses to human factor IX in factor IX knockout mice on BALB/c and C57BL/6 (B6) backgrounds, both strains having CD4+ T cells that proliferate in response to human factor IX. Surprisingly, wild-type mice have similar factor IX-recognizing CD4+ T cells. We defined a dominant CD4+ epitope for each strain (CVETGVKITVVAGEH for BALB/c and LLELDEPLVLNSYVTPIC for B6) that was recognized by both factor IX knockout and wild-type mice. While human factor IX did not cross-react with the mouse homologs of these epitopes, immunization with peptides from murine factor IX stimulated proliferation in factor IX knockout mice and wild-type mice, demonstrating a failure to delete murine factor IX-specific T cells in normal mice.     

Lentivirus-mediated platelet-derived factor VIII gene therapy in murine haemophilia A

BACKGROUND: Previous studies from our laboratory have demonstrated that lineage-targeted synthesis of factor VIII (FVIII) under the direction of the platelet-specific integrin alphaIIb gene promoter (2bF8) can correct the murine haemophilia A phenotype even in the presence of high titer inhibitory antibodies in a transgenic mouse model. OBJECTIVE: In this study, we assessed the efficacy of using a genetic therapy approach to correct haemophilia A in FVIII-deficient (FVIII(null)) mice by transplantation of bone marrow (BM) transduced with a lentivirus (LV)-based gene transfer cassette encoding 2bF8. RESULTS: Functional FVIII activity (FVIII:C) was detected in platelet lysates from treated mice and the levels were similar to 2bF8 heterozygous transgenic mice. Mice transplanted with 2bF8 LV-transduced BM survived tail clipping and we did not detected inhibitory or non-inhibitory FVIII antibodies over the period of this study (11 months). Furthermore, BM transferred from the primary transplant recipients into FVIII(null) secondary recipients demonstrated sustained platelet-FVIII expression leading to correction of the haemophilia A phenotype showing that gene transfer occurred within long-term repopulating haematopoietic stem cells. CONCLUSIONS: These results demonstrate that ectopic expression of FVIII in platelets by lentivirus-mediated bone marrow transduction/transplantation may be a promising strategy for gene therapy of haemophilia A in humans.     

Impact of the Underlying Mutation and the Route of Vector Administration on Immune Responses to Factor IX in Gene Therapy for Hemophilia B

Immune responses to factor IX (F.IX), a major concern in gene therapy for hemophilia, were analyzed for adeno-associated viral (AAV-2) gene transfer to skeletal muscle and liver as a function of the F9 underlying mutation. Vectors identical to those recently used in clinical trials were administered to four lines of hemophilia B mice on a defined genetic background [C3H/HeJ with deletion of endogenous F9 and transgenic for a range of nonfunctional human F.IX (hF.IX) variants]. The strength of the immune response to AAV-encoded F.IX inversely correlated with the degree of conservation of endogenous coding information and levels of endogenous antigen. Null mutation animals developed T- and B-cell responses in both protocols. However, inhibitor titers were considerably higher upon muscle gene transfer (or protein therapy). Transduced muscles of Null mice had strong infiltrates with CD8⁺ cells, which were much more limited in the liver and not seen for the other mutations. Sustained expression was achieved with liver transduction in mice with crm⁻ nonsense and missense mutations, although they still formed antibodies upon muscle gene transfer. Therefore, endogenous expression prevented T-cell responses more effectively than antibody formation, and immune responses varied substantially depending on the protocol and the underlying mutation.     

Muscle as a target for supplementary factor IX gene transfer

Immune responses to the factor IX (F.IX) transgene product are a concern in gene therapy for the X-linked bleeding disorder hemophilia B. The risk for such responses is determined by several factors, including the vector, target tissue, and others. Previously, we have demonstrated that hepatic gene transfer with adeno-associated viral (AAV) vectors can induce F.IX-specific immune tolerance. Muscle-derived F.IX expression, however, is limited by a local immune response. Here, skeletal muscle was investigated as a target for supplemental gene transfer. Given the low invasiveness of intramuscular injections, this route would be ideal for secondary gene transfer, thereby boosting levels of transgene expression. However, this is feasible only if immune tolerance established by compartmentalization of expression to the liver extends to other sites. Immune tolerance to human F.IX established by prior hepatic AAV-2 gene transfer was maintained after subsequent injection of AAV-1 or adenoviral vector into skeletal muscle, and tolerized mice failed to form antibodies or an interferon (IFN)-gamma(+) T cell response to human F.IX. A sustained increase in systemic transgene expression was obtained for AAV-1, whereas an increase after adenoviral gene transfer was transient. A CD8(+) T cell response specifically against adenovirus-transduced fibers was observed, suggesting that cytotoxic T cell responses against viral antigens were sufficient to eliminate expression in muscle. In summary, the data demonstrate that supplemental F.IX gene transfer to skeletal muscle does not break tolerance achieved by liver-derived expression. The approach is efficacious, if the vector for muscle gene transfer does not express immunogenic viral proteins.     

Hyperactive PiggyBac Transposons for Sustained and Robust Liver-targeted Gene Therapy

The development of robust nonviral vectors could facilitate clinical gene therapy applications and may overcome some of the immune complications of viral vectors. Nevertheless, most nonviral gene deliver approaches typically yield only transient and/or low gene expression. To address these caveats, we have explored piggyBac transposons to correct hemophilia B by liver-directed factor IX (FIX) gene therapy in hemophilic mice. To achieve this, we combined the use of: (i) a hyperactive codon-optimized piggyBac transposase, (ii) a computationally enhanced liver-specific promoter, (iii) a hyperfunctional codon-optimized FIX transgene (FIX R338L Padua), and (iv) a modification of the transposon terminal repeats. This combination strategy resulted in a robust 400-fold improvement in vector performance in hepatocytes, yielding stable supraphysiologic human FIX activity (>1 year). Liver-specific expression resulted in the induction of FIX-specific immune tolerance. Remarkably, only very low transposon/transposase doses were required to cure the bleeding diathesis. Similarly, PB transposons could be used to express supraphysiologic factor VIII levels using low transposon/transposase doses. PB transposition did not induce tumors in a sensitive hepatocellular carcinoma-prone mouse model. These results underscore the potency and relative safety of the latest generation PB transposons, which constitutes a versatile platform for stable and robust secretion of therapeutic proteins.     

Long-term transgene expression by administration of a lentivirus-based vector to the fetal circulation of immuno-competent mice

Inefficient gene transfer, inaccessibility of stem cell compartments, transient gene expression, and adverse immune and inflammatory reactions to vector and transgenic protein are major barriers to successful in vivo application of gene therapy for most genetic diseases. Prenatal gene therapy with integrating vectors may overcome these problems and prevent early irreparable organ damage. To this end, high-dose attenuated VSV-G pseudotyped equine infectious anaemia virus (EIAV) encoding beta-galactosidase under the CMV promoter was injected into the fetal circulation of immuno-competent MF1 mice. We saw prolonged, extensive gene expression in the liver, heart, brain and muscle, and to a lesser extent in the kidney and lung of postnatal mice. Progressive clustered hepatocyte staining suggests clonal expansion of cells stably transduced. We thus provide proof of principle for efficient gene delivery and persistent transgene expression after prenatal application of the EIAV vector and its potential for permanent correction of genetic diseases.