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Gene therapy (GT) is becoming a realistic treatment option for patients with haemophilia. Outside clinical trials, the complexity and potential complications of GT will pose unprecedented challenges to haemophilia care centres.AIM: To explore the potential use of electronic tools to improve the delivery of GT under real-world conditions.METHODS: Considering the hub-and-spoke model, the GTH working group on GT considered the entire patient pathway and reached consensus on requirements for an integrative software tool to secure documenting and sharing information between treaters, pharmacies and patients.RESULTS: Six steps of the gene therapy process were identified, each requiring completion of the previous step as a prerequisite for entry. The responsibilities of GT dosing and follow-up treatment centres, read/write access rules, and the minimum data set were outlined. Data contributed by patients through mobile devices was also considered.CONCLUSION: Important information needs to be shared between patients and treatment centres in a real-world GT hub-and-spoke model. Collecting and sharing this information in well-organised electronic applications will not only improve patient care but also enable national and international data collection in clinical registries...
The physiotherapist plays an essential role for people with haemophilia, an inherited bleeding disease responsible for musculoskeletal complications. Yet, with the advent of new and advanced therapies, the medical landscape is changing, and physiotherapy must adapt alongside. This paper considers whether there will still be a need for physiotherapy in the era of advanced therapies, and discusses ways in which services should evolve to complement emerging treatment paradigms for haemostasis in people with haemophilia. Ultimately, physiotherapy will remain an important element of care, even for people with little joint damage and low risks in the era of the new mild phenotype. However, competencies will need to evolve, and physiotherapists in both primary care and specialist treatment centres should work with haematology colleagues to develop more sensitive tools for detecting early joint changes. Physiotherapists will also play a crucial role in counselling and physically coaching, monitoring the musculoskeletal status of people with haemophilia who have transitioned to new treatments.
Introduction: Adeno-associated virus (AAV)-based gene therapy for haemophilia presents a challenge to the existing structure of haemophilia centres and requires a rethink of current collaboration and information exchange with the aim of ensuring a system that is fit-for-purpose for advanced therapies to maximise benefits and minimise risks. In Europe, a certification process based on the number of patients and facilities is offered to the haemophilia centres by European Haemophilia Network (EUHANET). Aim and methods: This joint European Association for Haemophilia and Allied Disorders (EAHAD) and European Haemophilia Consortium (EHC) publication describes criteria for centres participating in gene therapy care that require a reassessment of the infrastructure of comprehensive care and provides an outlook on how these criteria can be implemented in the future work of haemophilia centres. Results: The core definition of a haemophilia treatment centre remains, but additional roles could be implemented. A modifiable ‘hub-and-spoke’ model addresses all aspects associated with gene therapy, including preparation and administration of the gene therapy product, determination of coagulation and immunological parameters, joint score and function, and liver health. This will also include the strategy on how to follow-up patients for a long-term safety and efficacy surveillance. Conclusion: We propose a modifiable, networked ‘hub and spoke’ model with a long term safety and efficacy surveillance system. This approach will be progressively developed with the goal of making haemophilia centres better qualified to deliver gene therapy and to make gene therapy accessible to all persons with haemophilia, irrespective of their country or centre of origin.
Introduction: Adeno-associated virus (AAV)-based gene therapy for haemophilia presents a challenge to the existing structure of haemophilia centres and requires a rethink of current collaboration and information exchange with the aim of ensuring a system that is fit-for-purpose for advanced therapies to maximise benefits and minimise risks. In Europe, a certification process based on the number of patients and facilities is offered to the haemophilia centres by European Haemophilia Network (EUHANET). Aim and methods: This joint European Association for Haemophilia and Allied Disorders (EAHAD) and European Haemophilia Consortium (EHC) publication describes criteria for centres participating in gene therapy care that require a reassessment of the infrastructure of comprehensive care and provides an outlook on how these criteria can be implemented in the future work of haemophilia centres. Results: The core definition of a haemophilia treatment centre remains, but additional roles could be implemented. A modifiable ‘hub-and-spoke’ model addresses all aspects associated with gene therapy, including preparation and administration of the gene therapy product, determination of coagulation and immunological parameters, joint.
Autologous chimeric antigen receptor-modified (CAR) T cells with specificity for CD19 showed potent antitumor efficacy in clinical trials against relapsed and refractory B-cell acute lymphoblastic leukemia (B-ALL). Contrary to T cells, natural killer (NK) cells kill their targets in a non-antigen-specific manner and do not carry the risk of inducing graft vs. host disease (GvHD), allowing application of donor-derived cells in an allogenic setting. Hence, unlike autologous CAR-T cells, therapeutic CD19-CAR-NK cells can be generated as an off-the-shelf product from healthy donors. Nevertheless, genetic engineering of peripheral blood (PB) derived NK cells remains challenging and optimized protocols are needed. In our study, we aimed to optimize the generation of CD19-CAR-NK cells by retroviral transduction to improve the high antileukemic capacity of NK cells. We compared two different retroviral vector platforms, the lentiviral and alpharetroviral, both in combination with two different transduction enhancers (Retronectin and Vectofusin-1). We further explored different NK cell isolation techniques (NK cell enrichment and CD3/CD19 depletion) to identify the most efficacious methods for genetic engineering of NK cells. Our results demonstrated that transduction of NK cells with RD114-TR pseudotyped retroviral vectors, in combination with Vectofusin-1 was the most efficient method to generate CD19-CAR-NK cells. Retronectin was potent in enhancing lentiviral/VSV-G gene delivery to NK cells but not alpharetroviral/RD114-TR. Furthermore, the Vectofusin-based transduction of NK cells with CD19-CARs delivered by alpharetroviral/RD114-TR and lentiviral/RD114-TR vectors outperformed lentiviral/VSV-G vectors. The final generated CD19-CAR-NK cells displayed superior cytotoxic activity against CD19-expressing target cells when compared to non-transduced NK cells achieving up to 90% specific killing activity. In summary, our findings present the use of RD114-TR pseudotyped retroviral particles in combination with Vectofusin-1 as a successful strategy to genetically modify PB-derived NK cells to achieve highly cytotoxic CD19-CAR-NK cells at high yield.
Gene therapy has the potential to revolutionise treatment for patients with haemophilia and is close to entering clinical practice. While factor concentrates have improved outcomes, individuals still face a lifetime of injections, pain, progressive joint damage, the potential for inhibitor development and impaired quality of life. Recently published studies in adeno‐associated viral (AAV) vector‐mediated gene therapy have demonstrated improvement in endogenous factor levels over sustained periods, significant reduction in annualised bleed rates, lower exogenous factor usage and thus far a positive safety profile. In making the shared decision to proceed with gene therapy for haemophilia, physicians should make it clear that research is ongoing and that there are remaining evidence gaps, such as long‐term safety profiles and duration of treatment effect. The eligibility criteria for gene therapy trials mean that key patient groups may be excluded, eg children/adolescents, those with liver or kidney dysfunction and those with a prior history of factor inhibitors or pre‐existing neutralising AAV antibodies. Gene therapy offers a life‐changing opportunity for patients to reduce their bleeding risk while also reducing or abrogating the need for exogenous factor administration. Given the expanding evidence base, both physicians and patients will need sources of clear and reliable information to be able to discuss and judge the risks and benefits of treatment.
The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform that combines simplicity, inexpensive manufacture, and favorable safety features in the context of human applications. However, efficient correction of hematopoietic stem and progenitor cells (HSPCs) with non-viral vector systems, including SB, demands further refinement of gene delivery techniques. We set out to improve SB gene transfer into hard-to-transfect human CD34+ cells by vectorizing the SB system components in the form of minicircles that are devoid of plasmid backbone sequences and are, therefore, significantly reduced in size. As compared to conventional plasmids, delivery of the SB transposon system as minicircle DNA is ∼20 times more efficient, and it is associated with up to a 50% reduction in cellular toxicity in human CD34+ cells. Moreover, providing the SB transposase in the form of synthetic mRNA enabled us to further increase the efficacy and biosafety of stable gene delivery into hematopoietic progenitors ex vivo. Genome-wide insertion site profiling revealed a close-to-random distribution of SB transposon integrants, which is characteristically different from gammaretroviral and lentiviral integrations in HSPCs. Transplantation of gene-marked CD34+ cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution, which was most efficient when the SB transposase was supplied as mRNA and nucleofected cells were maintained for 4–8 days in culture before transplantation. Collectively, implementation of minicircle and mRNA technologies allowed us to further refine the SB transposon system in the context of HSPC gene delivery to ultimately meet clinical demands of an efficient and safe non-viral gene therapy protocol.
Ocular gene therapy approaches have been developed for a variety of different diseases. In particular, clinical gene therapy trials for RPE65 mutations, X-linked retinoschisis, and choroideremia have been conducted at different centers in recent years, showing that adeno-associated virus (AAV)-mediated gene therapy is safe, but limitations exist as to the therapeutic benefit and long-term duration of the treatment. The technique of vector delivery to retinal cells relies on subretinal injection of the vector solution, causing a transient retinal detachment. Although retinal detachments are known to cause remodeling of retinal neuronal structures as well as significant cell loss, the possible effects of this short-term therapeutic retinal detachment on retinal structure and circuitry have not yet been studied in detail. In this study, retinal morphology and apoptotic status were examined in healthy rat retinas following AAV-mediated gene transfer via subretinal injection with AAV2/5.CMV.d2GFP or sham injection with fluorescein. Outer plexiform layer (OPL) morphology was assessed by immunohistochemical labeling, laser scanning confocal microscopy, and electron microscopy. The number of synaptic contacts in the OPL was quantified after labeling with structural markers. To assess the apoptotic status, inflammatory and pro-apoptotic markers were tested and TUNEL assay for the detection of apoptotic nuclei was performed. Pre- and postsynaptic structures in the OPL, such as synaptic ribbons or horizontal and bipolar cell processes, did not differ in size or shape in injected versus non-injected areas and control retinas. Absolute numbers of synaptic ribbons were not altered. No signs of relevant gliosis were detected. TUNEL labeling of retinal cells did not vary between injected and non-injected areas, and apoptosis-inducing factor was not delocalized to the nucleus in transduced areas. The neuronal circuits in the OPL of healthy rat retinas undergoing AAV-mediated gene transfer were not altered by the temporary retinal detachment caused by subretinal injection, the presence of viral particles, or the expression of green fluorescent protein as a transgene. This observation likely requires further investigations in the dog model for RPE65 deficiency in order to determine the impact of RPE65 transgene expression on diseased retinas in animals and men.
T-cell receptor (TCR) polyclonal mature T cells are surprisingly resistant to oncogenic transformation after retroviral insertion of T-cell oncogenes. In a mouse model, it has been shown that mature T-cell lymphoma/leukemia (MTCLL) is not induced upon transplantation of mature, TCR polyclonal wild-type (WT) T cells, transduced with gammaretroviral vectors encoding potent T-cell oncogenes, into RAG1-deficient recipients. However, further studies demonstrated that quasi-monoclonal T cells treated with the same protocol readily induced MTCLL in the recipient mice. It has been hypothesized that in the TCR polyclonal situation, outgrowth of preleukemic cells and subsequent conversion to overt malignancy is suppressed through regulation of clonal abundances on a per-clone basis due to interactions between TCRs and self-peptide-MHC-complexes (spMHCs), while these mechanisms fail in the quasi-monoclonal situation. To quantitatively study this hypothesis, we applied a mathematical modeling approach. In particular, we developed a novel ordinary differential equation model of T-cell homeostasis, in which T-cell fate depends on spMHC-TCR-interaction-triggered stimulatory signals from antigen-presenting cells (APCs). Based on our mathematical modeling approach, we identified parameter configurations of our model, which consistently explain the observed phenomena. Our results suggest that the preleukemic cells are less competent than healthy competitor cells in acquiring survival stimuli from APCs, but that proliferation of these preleukemic cells is less dependent on survival stimuli from APCs. These predictions now call for experimental validation.
In vivo manipulation of interleukin-2 expression by a retroviral tetracycline (tet)-regulated system
(1999)
We have used the tetracycline (tet)-regulated system as described previously to evaluate the applicability of controlled gene expression in cancer gene therapy. As a model gene, we used the human interleukin-2 (IL-2) gene, which has been placed under the transcriptional control of the tetO/promoter. Human melanoma cells were transduced by two modified retroviral tet vectors containing the transactivator regulatory unit and the IL-2 gene driven by the tetO/promoter, respectively. In the absence of tet, IL-2 expression in the target cells was stable over several months. IL-2 production was in the range of 40 U/106 cells/24 hours. A fine tuning of IL-2 expression could be achieved by culturing the transduced cells with increasing doses of tet, whereby a concentration of 500 ng/mL tet in the culture medium abrogated IL-2 expression. Most importantly for clinical application, IL-2 expression by the transduced melanoma cells could also be regulated in vivo. When nu/nu mice were inoculated with the transduced tumor cells, they failed to develop tumors. Instead, the inhibition of IL-2 expression in the transduced tumor cells by oral administration of tet led to subcutaneous tumor growth; this growth rate was comparable with the growth rate of subcutaneously inoculated untransduced parental cells. The finding demonstrates the applicability of the tet-regulated system in cancer gene therapy.