Promoterless gene targeting without nucleases ameliorates haemophilia B in mice.

Site-specific gene addition can allow stable transgene expression for gene therapy. When possible, this is preferred over the use of promiscuously integrating vectors, which are sometimes associated with clonal expansion and oncogenesis. Site-specific endonucleases that can induce high rates of targeted genome editing are finding increasing applications in biological discovery and gene therapy. However, two safety concerns persist: endonuclease-associated adverse effects, both on-target and off-target; and oncogene activation caused by promoter integration, even without nucleases. Here we perform recombinant adeno-associated virus (rAAV)-mediated promoterless gene targeting without nucleases and demonstrate amelioration of the bleeding diathesis in haemophilia B mice. In particular, we target a promoterless human coagulation factor IX (F9) gene to the liver-expressed mouse albumin (Alb) locus. F9 is targeted, along with a preceding 2A-peptide coding sequence, to be integrated just upstream to the Alb stop codon. While F9 is fused to Alb at the DNA and RNA levels, two separate proteins are synthesized by way of ribosomal skipping. Thus, F9 expression is linked to robust hepatic albumin expression without disrupting it. We injected an AAV8-F9 vector into neonatal and adult mice and achieved on-target integration into ∼0.5% of the albumin alleles in hepatocytes. We established that F9 was produced only from on-target integration, and ribosomal skipping was highly efficient. Stable F9 plasma levels at 7-20% of normal were obtained, and treated F9-deficient mice had normal coagulation times. In conclusion, transgene integration as a 2A-fusion to a highly expressed endogenous gene may obviate the requirement for nucleases and/or vector-borne promoters. This method may allow for safe and efficacious gene targeting in both infants and adults by greatly diminishing off-target effects while still providing therapeutic levels of expression from integration.

Efficient lentiviral transduction of liver requires cell cycling in vivo.

Human-immunodeficiency-virus (HIV)-based lentiviral vectors are a promising tool for in vivo gene therapy. Unlike Moloney-murine-leukaemia-based retroviruses (MLV), lentiviruses are believed to stably transduce quiescent (non-cycling) cells in various organs. No previous studies, however, have directly established the cell-cycle status of any transduced cell type at the time of vector administration in vivo. In vitro studies using wild-type HIV or HIV-based vectors have shown that, in some cases, cell-cycle activation is required for infection, even though cellular mitosis is not an absolute requirement for integration. Even if the block in reverse transcription is overcome in quiescent T cells, productive infection by HIV cannot be rescued in the absence of cell-cycle activation. The potential use of these vectors for gene therapy prompted our study, which establishes a cell-cycle requirement for efficient transduction of hepatocytes in vivo.

Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system.

The development of non-viral gene-transfer technologies that can support stable chromosomal integration and persistent gene expression in vivo is desirable. Here we describe the successful use of transposon technology for the nonhomologous insertion of foreign genes into the genomes of adult mammals using naked DNA. We show that the Sleeping Beauty transposase can efficiently insert transposon DNA into the mouse genome in approximately 5-6% of transfected mouse liver cells. Chromosomal transposition resulted in long-term expression (>5 months) of human blood coagulation factor IX at levels that were therapeutic in a mouse model of haemophilia B. Our results establish DNA-mediated transposition as a new genetic tool for mammals, and provide new strategies to improve existing non-viral and viral vectors for human gene therapy applications.

Long-term hepatic adenovirus-mediated gene expression in mice following CTLA4Ig administration.

Recombinant adenovirus vectors are efficient at transferring genes into somatic tissues but are limited for use in clinical gene therapy by immunologic factors that result in the rapid loss of gene expression and inhibit secondary gene transfer. This study demonstrates that systemic coadministration of recombinant adenovirus with soluble CTLA4Ig, which is known to block co-stimulatory signals between T cells and antigen presenting cells, leads to persistent adenoviral gene expression in mice without long-term immunosuppression. This form of immunotherapy greatly enhances the likelihood that recombinant adenovirus vectors will be useful for human gene therapy.

Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors.

Haemophilia B, or factor IX deficiency, is a X-linked recessive disorder that occurs in about one in 25,000 males, and severely affected people are at risk for spontaneous bleeding into numerous organs. Bleeding can be life-threatening or lead to chronic disabilities with haemophilic arthropathy. The severity of the bleeding tendency varies among patients and is related to the concentration of functional plasma factor IX. Patients with 5-30% of the normal factor IX have mild haemophilia that may not be recognized until adulthood or after heavy trauma or surgery. Therapy for acute bleeding consists of the transfusion of clotting-factor concentrates prepared from human blood and recombinant clotting factors that are currently in clinical trials. Both recombinant retroviral and adenoviral vectors have successfully transferred factor IX cDNA into the livers of dogs with haemophilia B. Recombinant retroviral-mediated gene transfer results in persistent yet subtherapeutic concentrations of factor IX and requires the stimulation of hepatocyte replication before vector administration. Recombinant adenoviral vectors can temporarily cure the coagulation defect in the canine haemophilia B model; however, an immune response directed against viral gene products made by the vector results in toxicity and limited gene expression. The use of recombinant adeno-associated virus (rAAV) vectors is promising because the vector contains no viral genes and can transduce non-dividing cells. The efficacy of in vivo transduction of non-dividing cells has been demonstrated in a wide variety of tissues. In this report, we describe the successful transduction of the liver in vivo using r-AAV vectors delivered as a single administration to mice and demonstrate that persistent, curative concentrations of functional human factor IX can be achieved using wild-type-free and adenovirus-free rAAV vectors. This demonstrates the potential of treating haemophilia B by gene therapy at the natural site of factor IX production.

Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector.

Pre-clinical studies in mice and haemophilic dogs have shown that introduction of an adeno-associated viral (AAV) vector encoding blood coagulation factor IX (FIX) into skeletal muscle results in sustained expression of F.IX at levels sufficient to correct the haemophilic phenotype. On the basis of these data and additional pre-clinical studies demonstrating an absence of vector-related toxicity, we initiated a clinical study of intramuscular injection of an AAV vector expressing human F.IX in adults with severe haemophilia B. The study has a dose-escalation design, and all patients have now been enrolled in the initial dose cohort (2 x 10(11) vg/kg). Assessment in the first three patients of safety and gene transfer and expression show no evidence of germline transmission of vector sequences or formation of inhibitory antibodies against F.IX. We found that the vector sequences are present in muscle by PCR and Southern-blot analyses of muscle biopsies and we demonstrated expression of F.IX by immunohistochemistry. We observed modest changes in clinical endpoints including circulating levels of F.IX and frequency of FIX protein infusion. The evidence of gene expression at low doses of vector suggests that dose calculations based on animal data may have overestimated the amount of vector required to achieve therapeutic levels in humans, and that the approach offers the possibility of converting severe haemophilia B to a milder form of the disease.

The effect of physical activity on mother-child relationship and parental attitudes: a follow-up study examining the long-term effects of COVID-19.

OBJECTIVE: This study was planned to evaluate effects of game-based physical activity model on mother-child relationship and parental attitudes during the prolonged COVID-19 pandemic period. SUBJECTS AND METHODS: This study was designed using a web-based quasi-experimental model with a pre-test/post-test evaluation, with a control group. The mothers who accepted to participate in the study and their children were divided into experimental (group I, n=28) and control groups (group II, n=31). The mothers and children in the experimental group were asked to apply web-based game-based physical activity model for 20 minutes/day for 4 weeks. The online questionnaire included socio-demographic data form, Child Parent Relationship Scale (CPRS), and Parental Attitude Scale (PAS). RESULTS: There were no significant differences between mean scores of pre-test and post-test subscales of the PAS in group I (p>0.05 for all subscales). It was found that post-test scores of democratic subscales of PAS statistically significant decreased (p=0.047) and the authoritarian attitude subscale scores significantly increased (p=0.033) in group II. The mean pre- and post-activity scores of positive/close relationship and conflictual relationship subscales of CPRS differ between groups (p<0.05 for both subscales). Pre-post test scores of group II were found to be significantly lower compared to group II. CONCLUSIONS: Our study provides a moderate improvement in parameters evaluated; however, we suggest that longer-term activities may have a more permanent and statistically significant effect.

Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics.

Considered by some to be among the simpler forms of life, viruses represent highly evolved natural vectors for the transfer of foreign genetic information into cells. This attribute has led to extensive attempts to engineer recombinant viral vectors for the delivery of therapeutic genes into diseased tissues. While substantial progress has been made, and some clinical successes are over the horizon, further vector refinement and/or development is required before gene therapy will become standard care for any individual disorder.

Sustained survival of human hepatocytes in mice: A model for in vivo infection with human hepatitis B and hepatitis delta viruses.

Persistence of hepatocytes transplanted into the same or related species has been established. The long-term engraftment of human hepatocytes into rodents would be useful for the study of human viral hepatitis, where it might allow the species, technical and size limitations of the current animal models to be overcome. Although transgenic mice expressing the hepatitis B virus (HBV) genome produce infectious virus in their serum, the viral life cycle is not complete, in that the early stages of viral binding and entry into hepatocytes and production of an episomal transcriptional DNA template do not occur. As for hepatitis delta virus (HDV), another cause of liver disease, no effective therapy exists to eradicate infection, and it remains resistant even to recent regimens that have considerably changed the treatment of HBV (ref. 13). Here, we demonstrate long-term engraftment of primary human hepatocytes transplanted in a matrix under the kidney capsule of mice with administration of an agonistic antibody against c-Met. These mice were susceptible to HBV infection and completion of the viral life cycle. In addition, we demonstrate super-infection of the HBV-infected mice with HDV. Our results describe a new xenotransplant model that allows study of multiple aspects of human hepatitis viral infections, and may enhance studies of human liver diseases.

Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors.

Hemophilia B, or factor IX deficiency, is an X-linked recessive disorder occurring in about 1 in 25,000 males. Affected individuals are at risk for spontaneous bleeding into many organs; treatment mainly consists of the transfusion of clotting factor concentrates prepared from human blood or recombinant sources after bleeding has started. Small- and large-animal models have been developed and/or characterized that closely mimic the human disease state. As a preclinical model for gene therapy, recombinant adeno-associated viral vectors containing the human or canine factor IX cDNAs were infused into the livers of murine and canine models of hemophilia B, respectively. There was no associated toxicity with infusion in either animal model. Constitutive expression of factor IX was observed, which resulted in the correction of the bleeding disorder over a period of over 17 months in mice. Mice with a steady-state concentration of 25% of the normal human level of factor IX had normal coagulation. In hemophilic dogs, a dose of rAAV that was approximately 1/10 per body weight that given to mice resulted in 1% of normal canine factor IX levels, the absence of inhibitors, and a sustained partial correction of the coagulation defect for at least 8 months.

Prevention of spontaneous bleeding in dogs with haemophilia A and haemophilia B.

Dogs with haemophilia A or haemophilia B exhibit spontaneous bleeding comparable with the spontaneous bleeding phenotype that occurs in humans with severe haemophilia. The phenotypic and genotypic characteristics of haemophilic dogs have been well-described, and such dogs are suitable for testing prophylactic protein replacement therapy and gene transfer strategies. In dogs with haemophilia, long-term effects on spontaneous bleeding frequency (measured over years) can be used as an efficacy endpoint in such studies. Although complete correction of coagulopathy has not been achieved, published data show that prophylactic factor replacement therapy and gene transfer can markedly reduce the frequency of spontaneous bleeding in haemophilic dogs. Further studies are currently ongoing.

In vivo gene therapy of hemophilia B: sustained partial correction in factor IX-deficient dogs.

The liver represents a model organ for gene therapy. A method has been developed for hepatic gene transfer in vivo by the direct infusion of recombinant retroviral vectors into the portal vasculature, which results in the persistent expression of exogenous genes. To determine if these technologies are applicable for the treatment of hemophilia B patients, preclinical efficacy studies were done in a hemophilia B dog model. When the canine factor IX complementary DNA was transduced directly into the hepatocytes of affected dogs in vivo, the animals constitutively expressed low levels of canine factor IX for more than 5 months. Persistent expression of the clotting factor resulted in reductions of whole blood clotting and partial thromboplastin times of the treated animals. Thus, long-term treatment of hemophilia B patients may be feasible by direct hepatic gene therapy in vivo.

Gene therapy for metabolic disorders.

Most diseases caused by genetic deficiencies could, in theory, be treated by the introduction and expression of a normal gene into an appropriate target tissue. It seems likely that gene therapy strategies for most metabolic disorders will not require strict gene regulation, as a fraction of the normal levels of gene activity could result in amelioration or significant improvement in the clinical outcome. Gene therapy is making rapid progress towards the goal of treating various disorders: here, we summarize the state of gene therapy for metabolic disorders.

Muscle differentiation during repair of myocardial necrosis in rats via gene transfer with MyoD.

Myocardial infarcts heal by scar formation because there are no stem cells in myocardium, and because adult myocytes cannot divide and repopulate the wound. We sought to redirect the heart to form skeletal muscle instead of scar by transferring the myogenic determination gene, MyoD, into cardiac granulation (wound repair) tissue. A replication-defective adenovirus was constructed containing MyoD under transcriptional control of the Rous sarcoma virus long terminal repeat. The virus converted cultured cardiac fibroblasts to skeletal muscle, indicated by expression of myogenin and skeletal myosin heavy chains (MHCs). To determine if MyoD could induce muscle differentiation in vivo, we injected 2 x 10(9) or 10(10) pfu of either the MyoD or a control beta-galactosidase adenovirus into healing rat hearts, injured 1 wk previously by freeze-thaw. After receiving the lower viral dose, cardiac granulation tissue expressed MyoD mRNA and protein, but did not express myogenin or skeletal MHC. When the higher dose of virus was administered, double immunostaining showed that cells in reparative tissue expressed both myogenin and embryonic skeletal MHC. No muscle differentiation occurred after beta-galactosidase transfection. Thus, MyoD gene transfer can induce skeletal muscle differentiation in healing heart lesions. Modifications of this strategy might eventually provide new contractile tissue to repair myocardial infarcts.

Selective translational regulation of ribosomal protein gene expression during early development of Drosophila melanogaster.

We have previously characterized a cloned cDNA coding for a developmentally regulated mRNA in Drosophila melanogaster whose expression is selectively regulated at the translational level during oogenesis and embryogenesis. In this report we show that this translationally regulated mRNA (rpA1) codes for an acidic ribosomal protein. Furthermore, our results indicate that most ribosomal protein mRNAs are regulated similarly to rpA1 mRNA. This conclusion is based on cell-free translation of mRNAs derived from polysomes and postpolysomal supernatants as well as in vivo labeling experiments. Thus, the translation of many ribosomal protein mRNAs appears to be temporally related to the synthesis of rRNA during D. melanogaster development. The relationship between rRNA transcription and ribosomal protein mRNA translation was further investigated by genetically reducing rRNA synthesis with the use of bobbed mutants. Unexpectedly, neither ribosomal protein mRNA abundance nor translation was altered in these mutants.

Increasing the size of rAAV-mediated expression cassettes in vivo by intermolecular joining of two complementary vectors.

A major shortcoming to the use of adeno-associated virus (rAAV) vectors is their limited packaging size. To overcome this hurdle, we split an expression cassette and cloned it into two separate vectors. The vectors contained either a nuclear localizing Escherichia coli lacZ transgene (nlslacZ) with a splice acceptor, or the human elongation factor 1alpha ( EF1alpha) gene enhancer/promoter(s) (EF1alphaEP) with a splice donor. We co-injected a promoter-less nlslacZ vector with a vector containing either a single EF1alphaEP or a double copy of the EF1alphaEP in a head-to-head orientation, into the portal vein of mice. Gene expression, measured by both transduction efficiency and quantitation of the recombinant protein, was as much as 60-70% of that obtained from mice that received a single vector containing a complete EFalphaEP/nlslacZ expression cassette. This two-vector approach may allow development of gene therapy strategies that will carry exogenous DNA sequences with large therapeutic cDNAs and/or regulatory elements.

Adenoviral preterminal protein stabilizes mini-adenoviral genomes in vitro and in vivo.

In the absence of host immunity, nonintegrating, first-generation adenoviral vectors remain stable in the nucleus of quiescent transduced cells in mice. A mini-adenoviral genome (9 kb) deleted for viral E1, E2, E3, and late genes, but containing the viral inverted terminal repeats (ITRs), transgene expression cassette (human alpha 1-antitrypsin), and the viral E4 genes was equally efficient at transducing cells in vitro or in vivo as first generation, E1-deleted vectors. In contrast to a first generation vector, gene expression as well as vector DNA was short-lived in cells transduced with the deleted adenoviral genome. We demonstrate that coexpression of the adenoviral E2-preterminal protein from the vector or in trans stabilizes the mini-genome in vitro and in vivo without evidence of cellular toxicity.

Hepatocyte transplantation: clinical and experimental application.

Hepatocyte transplantation has been proposed as an alternative to whole-organ transplantation to support many forms of hepatic insufficiency. Based on a significant body of work, the technique of hepatocyte transplantation has recently moved into the clinic in order to reestablish liver function without organ transplantation or to bridge the time between whole-organ liver transplantation. In addition, hepatocyte transplantation has also been proposed as a liver-directed gene therapy for a number of inherited hepatic disorders by transplanting either freshly isolated hepatocytes or genetically altered hepatocytes. To establish a research system based on the developing technology of hepatocyte transplantation, chimeric small animal models using human hepatocytes have recently been established, which would allow the study of human hepatocyte-specific functions, such as hepatitis viral infection and replication in vivo. Various aspects related to the recent progress and existing obstacles in the area of hepatocyte transplantation are summarized in this report.