The WOEST 2 registry : A prospective registry on antithrombotic therapy in atrial fibrillation patients undergoing percutaneous coronary intervention.

BACKGROUND: Patients on oral anticoagulants (OACs) undergoing percutaneous coronary intervention (PCI) also require aspirin and a P2Y12 inhibitor (triple therapy). However, triple therapy increases bleeding. The use of non-vitamin K antagonist oral anticoagulants (NOACs) and stronger P2Y12 inhibitors has increased. The aim of our study was to gain insight into antithrombotic management over time. METHODS: A prospective cohort study of patients on OACs for atrial fibrillation or a mechanical heart valve undergoing PCI was performed. Thrombotic outcomes were myocardial infarction, stroke, target-vessel revascularisation and all-cause mortality. Bleeding outcome was any bleeding. We report the 30-day outcome. RESULTS: The mean age of the 758 patients was 73.5 ± 8.2 years. The CHA2DS2-VASc score was ≥ 3 in 82% and the HAS-BLED score ≥ 3 in 44%. At discharge, 47% were on vitamin K antagonists (VKAs), 52% on NOACs, 43% on triple therapy and 54% on dual therapy. Treatment with a NOAC plus clopidogrel increased from 14% in 2014 to 67% in 2019. The rate of thrombotic (4.5% vs 2.0%, p = 0.06) and bleeding (17% vs. 14%, p = 0.42) events was not significantly different in patients on VKAs versus NOACs. Also, the rate of thrombotic (2.9% vs 3.4%, p = 0.83) and bleeding (18% vs 14%, p = 0.26) events did not differ significantly between patients on triple versus dual therapy. CONCLUSIONS: Patients on combined oral anticoagulation and antiplatelet therapy undergoing PCI are elderly and have both a high bleeding and ischaemic risk. Over time, a NOAC plus clopidogrel became the preferred treatment. The rate of thrombotic and bleeding events was not significantly different between patients on triple or dual therapy or between those on VKAs versus NOACs.

Next-generation muscle-directed gene therapy by in silico vector design.

There is an urgent need to develop the next-generation vectors for gene therapy of muscle disorders, given the relatively modest advances in clinical trials. These vectors should express substantially higher levels of the therapeutic transgene, enabling the use of lower and safer vector doses. In the current study, we identify potent muscle-specific transcriptional cis-regulatory modules (CRMs), containing clusters of transcription factor binding sites, using a genome-wide data-mining strategy. These novel muscle-specific CRMs result in a substantial increase in muscle-specific gene transcription (up to 400-fold) when delivered using adeno-associated viral vectors in mice. Significantly higher and sustained human micro-dystrophin and follistatin expression levels are attained than when conventional promoters are used. This results in robust phenotypic correction in dystrophic mice, without triggering apoptosis or evoking an immune response. This multidisciplinary approach has potentially broad implications for augmenting the efficacy and safety of muscle-directed gene therapy.

Haemophilia gene therapy: From trailblazer to gamechanger.

Haemophilia is an attractive disease target for gene therapy that fostered the development of the field at large. The delivery of the clotting factor genes into the patients' cells could be accomplished using different types of gene delivery vehicles or vectors. Adeno-associated viral vectors (AAV) and lentiviral vectors represent some of the most promising gene delivery technologies that allow for a relatively efficient delivery of the therapeutic FVIII and FIX transgenes into the relevant target cells. To reduce the risks associated with insertional mutagenesis due to random vector integration, gene-editing approaches have also been considered based primarily on zinc finger nuclease (ZFN) and CRISPR/Cas. However, comprehensive analysis of off-target effects is still required. It is particularly encouraging that relatively stable therapeutic FVIII or FIX expression levels were reached in severe haemophilia patients in recent clinical trials after liver-directed AAV gene therapy. This success could be ascribed in part to improvements in vector design. In particular, clotting factor levels could be increased by codon optimization of coagulation factor transgenes. Alternatively, incorporation of a hyperactive gain-of-function R338L mutation (FIX Padua) in the FIX gene improved the overall efficacy. However, some patients still show transient liver toxicity, especially at high vector doses, possibly due to inflammatory immune responses, requiring the need for transient immunosuppression. The exact immune mechanisms are not fully understood, but may at least in some patients involve an AAV-capsid specific T cell response. Moreover, there is a need to identify the key factors that contribute to the interpatient variability in therapeutic efficacy and safety after gene therapy.

Baboon envelope pseudotyped lentiviral vectors efficiently transduce human B cells and allow active factor IX B cell secretion in vivo in NOD/SCIDγc-/- mice.

Essentials B cells are attractive targets for gene therapy and particularly interesting for immunotherapy. A baboon envelope pseudotyped lentiviral vector (BaEV-LV) was tested for B-cell transduction. BaEV-LVs transduced mature and plasma human B cells with very high efficacy. BaEV-LVs allowed secretion of functional factor IX from B cells at therapeutic levels in vivo. SUMMARY: Background B cells are attractive targets for gene therapy for diseases associated with B-cell dysfunction and particularly interesting for immunotherapy. Moreover, B cells are potent protein-secreting cells and can be tolerogenic antigen-presenting cells. Objective Evaluation of human B cells for secretion of clotting factors such as factor IX (FIX) as a possible treatment for hemophilia. Methods We tested here for the first time our newly developed baboon envelope (BaEV) pseudotyped lentiviral vectors (LVs) for human (h) B-cell transduction following their adaptive transfer into an NOD/SCIDγc-/- (NSG) mouse. Results Upon B-cell receptor stimulation, BaEV-LVs transduced up to 80% of hB cells, whereas vesicular stomatitis virus G protein VSV-G-LV only reached 5%. Remarkably, BaEVTR-LVs permitted efficient transduction of 20% of resting naive and 40% of resting memory B cells. Importantly, BaEV-LVs reached up to 100% transduction of human plasmocytes ex vivo. Adoptive transfer of BaEV-LV-transduced mature B cells into NOD/SCID/γc-/- (NSG) [non-obese diabetic (NOD), severe combined immuno-deficiency (SCID)] mice allowed differentiation into plasmablasts and plasma B cells, confirming a sustained high-level gene marking in vivo. As proof of principle, we assessed BaEV-LV for transfer of human factor IX (hFIX) into B cells. BaEV-LVs encoding FIX efficiently transduced hB cells and their transfer into NSG mice demonstrated for the first time secretion of functional hFIX from hB cells at therapeutic levels in vivo. Conclusions The BaEV-LVs might represent a valuable tool for therapeutic protein secretion from autologous B cells in vivo in the treatment of hemophilia and other acquired or inherited diseases.

Percutaneous occlusion of post-myocardial infarction ventricular septum rupture.

AIMS: The aim of this systematic review is to gain insight into the published experience on percutaneous closure of a post-infarction ventricular septal rupture (VSR). METHOD: Relevant literature was obtained by MeSH-term searches in the online search-engine PubMed. Articles published in the last 10 years were included. Further filtering was done by using search limits and individual article selection based on the aims of this systematic review. CONCLUSION: Percutaneous closure is a potential technique in a select group of patients. The presence of cardiogenic shock and closure in the acute phase after VSR diagnosis are important risk factors of mortality. Device implantation is in general successful with few procedure-related complications. Reduction of the shunt fraction has been reported frequently. This technique is a less invasive alternative to surgical treatment and should be applied on a case-by-case basis.

Gene therapy for hemophilia.

Hemophilia A and B are X-linked monogenic disorders resulting from deficiencies of factor VIII and FIX, respectively. Purified clotting factor concentrates are currently intravenously administered to treat hemophilia, but this treatment is non-curative. Therefore, gene-based therapies for hemophilia have been developed to achieve sustained high levels of clotting factor expression to correct the clinical phenotype. Over the past two decades, different types of viral and non-viral gene delivery systems have been explored for hemophilia gene therapy research with a variety of target cells, particularly hepatocytes, hematopoietic stem cells, skeletal muscle cells, and endothelial cells. Lentiviral and adeno-associated virus (AAV)-based vectors are among the most promising vectors for hemophilia gene therapy. In preclinical hemophilia A and B animal models, the bleeding phenotype was corrected with these vectors. Some of these promising preclinical results prompted clinical translation to patients suffering from a severe hemophilic phenotype. These patients receiving gene therapy with AAV vectors showed long-term expression of therapeutic FIX levels, which is a major step forwards in this field. Nevertheless, the levels were insufficient to prevent trauma or injury-induced bleeding episodes. Another challenge that remains is the possible immune destruction of gene-modified cells by effector T cells, which are directed against the AAV vector antigens. It is therefore important to continuously improve the current gene therapy approaches to ultimately establish a real cure for hemophilia.

Alteration of cardiac progenitor cell potency in GRMD dogs.

Among the animal models of Duchenne muscular dystrophy (DMD), the Golden Retriever muscular dystrophy (GRMD) dog is considered the best model in terms of size and pathological onset of the disease. As in human patients presenting with DMD or Becker muscular dystrophies (BMD), the GRMD is related to a spontaneous X-linked mutation of dystrophin and is characterized by myocardial lesions. In this respect, GRMD is a useful model to explore cardiac pathogenesis and for the development of therapeutic protocols. To investigate whether cardiac progenitor cells (CPCs) isolated from healthy and GRMD dogs may differentiate into myocardial cell types and to test the feasibility of cell therapy for cardiomyopathies in a preclinical model of DMD, CPCs were isolated from cardiac biopsies of healthy and GRMD dogs. Gene profile analysis revealed an active cardiac transcription network in both healthy and GRMD CPCs. However, GRMD CPCs showed impaired self-renewal and cardiac differentiation. Population doubling and telomerase analyses highlighted earlier senescence and proliferation impairment in progenitors isolated from GRMD cardiac biopsies. Immunofluorescence analysis revealed that only wt CPCs showed efficient although not terminal cardiac differentiation, consistent with the upregulation of cardiac-specific proteins and microRNAs. Thus, the pathological condition adversely influences the cardiomyogenic differentiation potential of cardiac progenitors. Using PiggyBac transposon technology we marked CPCs for nuclear dsRed expression, providing a stable nonviral gene marking method for in vivo tracing of CPCs. Xenotransplantation experiments in neonatal immunodeficient mice revealed a valuable contribution of CPCs to cardiomyogenesis with homing differences between wt and dystrophic progenitors. These results suggest that cardiac degeneration in dystrophinopathies may account for the progressive exhaustion of local cardiac progenitors and shed light on cardiac stemness in physiological and pathological conditions. Furthermore, we provide essential information that canine CPCs may be used to alleviate cardiac involvement in a large preclinical model of DMD.

Relevance of an academic GMP Pan-European vector infra-structure (PEVI).

In the past 5 years, European investigators have played a major role in the development of clinical gene therapy. The provision of substantial funds by some individual member states to construct GMP facilities makes it an opportune time to network available gene therapy GMP facilities at an EU level. The integrated coordination of GMP production facilities and human skills for advanced gene and genetically-modified (GM) cell therapy, can dramatically enhance academic-led "First-in-man" gene therapy trials. Once proof of efficacy is gathered, technology can be transferred to the private sector which will take over further development taking advantage of knowledge and know-how. Complex technical challenges require existing production facilities to adapt to emerging technologies in a coordinated manner. These include a mandatory requirement for the highest quality of production translating gene-transfer technologies with pharmaceutical-grade GMP processes to the clinic. A consensus has emerged on the directions and priorities to adopt, applying to advanced technologies with improved efficacy and safety profiles, in particular AAV, lentivirus-based and oncolytic vectors. Translating cutting-edge research into "First-in-man" trials require that pre-normative research is conducted which aims to develop standard assays, processes and candidate reference materials. This research will help harmonise practices and quality in the production of GMP vector lots and GM-cells. In gathering critical expertise in Europe and establish conditions for interoperability, the PEVI infrastructure will contribute to the demands of the advanced therapy medicinal products* regulation and to both health and quality of life of EU-citizens.

Gene therapy, bioengineered clotting factors and novel technologies for hemophilia treatment.

The World Federation of Hemophilia estimates that of the 400,000 individuals worldwide with hemophilia, 300,000 receive either no, or very sporadic, treatment. Thus, considerable innovation will be required to provide cost-effective therapies/cures for all affected individuals. The high cost of prophylactic regimens hampers their widespread use, which further justifies the search for novel cost-effective therapies and ultimately a cure. Five gene transfer phase I clinical trials have been conducted using either direct in vivo gene delivery with viral vectors or ex vivo plasmid transfections and reimplantation of gene-engineered cells. Although there was evidence of gene transfer and therapeutic effects in some of these trials, stable expression of therapeutic factor VIII or FIX levels has not yet been obtained. Further improvements of the vectors and a better understanding of the immune consequences of gene transfer is warranted, as new trials are being initiated. Bioengineered clotting factors with increased stability and/or activity are being validated further in preclinical studies. Novel clotting factor formulations based on PEGylated liposomes with prolonged activities are being tested in the clinic, and are yielding encouraging results.

Efficacy and safety of adeno-associated viral vectors based on serotype 8 and 9 vs. lentiviral vectors for hemophilia B gene therapy.

BACKGROUND: Adeno-associated viral (AAV) and lentiviral vectors are promising vectors for gene therapy for hemophilia because they are devoid of viral genes and have the potential for long-term gene expression. OBJECTIVES: To compare the performance of different AAV serotypes (AAV8 and AAV9) vs. lentiviral vectors expressing factor (F) IX. METHODS AND RESULTS: AAV-based and lentiviral vectors were generated that express FIX from the same hepatocyte-specific expression cassette. AAV9 transduced the liver as efficiently as AAV8 and resulted in supra-physiological FIX levels (3000-6000% of normal) stably correcting the bleeding diathesis. Surprisingly, AAV9 resulted in unprecedented and widespread cardiac gene transfer, which was more efficient than with AAV8. AAV8 and AAV9 were not associated with any proinflammatory cytokine induction, in accordance with their minimal interactions with innate immune effectors. In contrast, lentiviral transduction resulted in modest and stable FIX levels near the therapeutic threshold (1%) and triggered a rapid self-limiting proinflammatory response (interleukin-6), which probably reflected their ability to efficiently interact with the innate immune system. CONCLUSIONS: AAV8 and 9 result in significantly higher FIX expression levels and have a reduced proinflammatory risk in comparison with lentiviral vectors. The unexpected cardiotropic properties of AAV9 have implications for gene therapy for heart disease.

Gene therapy for hemophilia "A" and "B": efficacy, safety and immune consequences.

The first successful gene therapy trials for the treatment of hereditary disorders underscore the potential of gene therapy to combat disease and alleviate human suffering. The development of gene therapy for hemophilia is not only a research priority in its own right but also serves as an ideal trailblazer for many different diseases. Significant progress has recently been made in the development of gene therapy for the treatment of hemophilia A and B. Long-term therapeutic levels of factor VIII and IX could be expressed following gene therapy in hemophilic mice, stably correcting the bleeding diathesis. These advances parallel the development of improved gene delivery systems. The induction of neutralizing antibodies (inhibitors) to the clotting factors could potentially preclude stable phenotypic correction. The risk of inhibitor formation varied, depending at least in part on the type of vector used and its in vivo tropism. We also demonstrated that the risk of immune responses to the vector particles, the clotting factors and/or transduced cells can be reduced by using vectors that only minimally interact with antigen presenting cells. In hemophilic mice, robust and stable clotting factor expression levels were achieved using adeno-associated viral vectors based on the newly disovered serotypes AAV8 and AAV9 which can efficient deliver the clotting factor genes into hepatocytes without triggering any inflammatory responses or adverse events. Pre-clinical studies in large animal models will be initiated to further validate these improved AAV vectors to ultimately justify a clinical trial in patients with severe hemophilia.

Gene therapy for haemophilia "A" and "B": efficacy, safety and immune consequences.

The first successful gene therapy trials for the treatment of hereditary disorders underscore the potential of gene therapy to combat disease and alleviate human suffering. The development of gene therapy for haemophilia is not only a research priority in its own right but also serves as an ideal trailblazer for many different diseases. Significant progress has recently been made in the development of gene therapy for the treatment of haemophilia A and B. Long-term therapeutic levels of factor VIII and IX could be expressed following gene therapy in haemophilic mice, stably correcting the bleeding diathesis. These advances parallel the development of improved gene delivery systems. The induction of neutralizing antibodies (inhibitors) to the clotting factors could potentially preclude stable phenotypic correction. The risk of inhibitor formation varied, depending at least in part on the type of vector used and its in vivo tropism. We also demonstrated that the risk of immune responses to the vector particles, the clotting factors and/or transduced cells can be reduced by using vectors that only minimally interact with antigen presenting cells. In haemophilic mice, robust and stable clotting factor expression levels were achieved using adeno-associated viral vectors based on the newly disovered serotypes AAV8 and AAV9 which can efficient deliver the clotting factor genes into hepatocytes without triggering any inflammatory responses or adverse events. Pre-clinical studies in large animal models will be initiated to further validate these improved AAV vectors to ultimately justify a clinical trial in patients with severe haemophilia.

Cellular and genetic therapies for haemophilia.

Haemophilia continues to be a prime target for a variety of gene and cell-based therapies. Pre-clinical successes in both mouse and dog models of the disease have been documented with a variety of approaches over the past decade, and there have now been six small clinical trials of gene transfer in haemophilia. To date, the only significant adverse events documented in these trials have been related to host immune responses, indicating that immunologic barriers continue to represent the major obstacle to achieving success of gene transfer in humans. Despite these challenges, new strategies are being explored with novel serotypes of viral vectors and with the use of transient periods of immunosuppression to attenuate the immune response to the vector and transgene product following gene delivery. Two new clinical trials, both using AAV vectors, will likely start within the next year, and additional large animal pre-clinical studies using other viral vector-mediated approaches for gene transfer are expected in the near future.

[Recent developments in gene therapy]. / Recente ontwikkelingen in gentherapie.

Gene therapy is defined as the introduction of genetic material in a patient's cells with resulting therapeutic benefit. It is a promising new biomedical discipline that could potentially lead to new treatments for hereditary diseases, cardiovascular and neurologic disorders, cancer, diabetes and even infectious diseases. The introduction of genetic material into somatic cells requires gene delivery vectors. Since viruses have developed efficient means to introduce their own genetic material into cells they can be readily adapted as viral vectors for gene therapy. Preclinical studies in animal models have shown that therapeutic effects can be achieved after gene therapy for genetic, acquired and complex disorders. Furthermore, therapeutic effects have been obtained in several phase I/II gene therapy clinical trials for hemophilia, severe combined immune deficiency (SCID) and cancer. Gene transfer technology has improved significantly over the past few years and has led to the development of vectors which have fewer side-effects without compromising their efficacy, at least partly due the development of cell-type specific targetable vectors. Nevertheless, the success of gene therapy is still very much depending upon the continuous development of improved vector technologies which would hopefully and ultimately cure diseases which are refractory to current treatment paradigms.

Preclinical and clinical gene therapy for haemophilia.

The goal of all haemophilia therapy is to prevent bleeding and its associated complications. Replacement by factor concentrates can only ever be suboptimum, and efforts are being made to correct the genetic cause of the disorder. Haemophilia is an ideal candidate for gene therapy, as it is caused by mutations in a single gene. A number of vectors have been used in an attempt to obtain therapeutic levels of factor VIII and factor IX in animal models, with some success. A number of phase 1 clinical trials have been conducted, and, although connection of the bleeding disorder was neither complete nor long-lasting, they do offer hope for a permanent gene-therapy cure for the disease.

Gene therapy for the hemophilias.

Significant progress has recently been made in the development of gene therapy for the treatment of hemophilia A and B. These advances parallel the development of improved gene delivery systems. Long-term therapeutic levels of factor (F) VIII and FIX can be achieved in adult FVIII- and FIX-deficient mice and in adult hemophiliac dogs using adeno-associated viral (AAV) vectors, high-capacity adenoviral vectors (HC-Ad) and lentiviral vectors. In mouse models, some of the highest FVIII or FIX expression levels were achieved using HC-Ad vectors with no or only limited adverse effects. Encouraging preclinical data have been obtained using AAV vectors, yielding long-term FIX levels above 10% in primates and in hemophilia B dogs, which prevented spontaneous bleeding. Non-viral ex vivo gene therapy approaches have also led to long-term therapeutic levels of coagulation factors in animal models. Nevertheless, the induction of neutralizing antibodies (inhibitors) to FVIII or FIX sometimes precludes stable phenotypic correction following gene therapy. The risk of inhibitor formation varies depending on the type of vector, vector serotype, vector dose, expression levels and promoter used, route of administration, transduced cell type and the underlying mutation in the hemophilia model. Some studies suggest that continuous expression of clotting factors may induce immune tolerance, particularly when expressed by the liver. Several gene therapy phase I clinical trials have been initiated in patients suffering from severe hemophilia A or B. Some subjects report fewer bleeding episodes and occasionally have low levels of clotting factor activity detected. Further improvement of the various gene delivery systems is warranted to bring a permanent cure for hemophilia one step closer to reality.

Bone marrow mesenchymal cells for haemophilia A gene therapy using retroviral vectors with modified long-terminal repeats.

Bone marrow (BM) cells are attractive target cells for ex vivo gene therapy of genetic diseases, including haemophilia A. However, BM-derived haematopoietic stem/progenitor cells (HSCs) transduced with factor VIII (FVIII) retroviral vectors, failed to express FVIII in vivo. To overcome the limitations of HSCs for haemophilia gene therapy, BM-derived mesenchymal cells were explored as alternative target cells. The BM mesenchymal cell population contains self-renewing mesenchymal stem/progenitor cells that give rise to different mesenchymal lineages and have been used safely in phase I gene-marking trials. Human BM mesenchymal cells were transduced in vitro with an improved retroviral vector encoding a human B-domain deleted FVIII (hFVIIIdeltaB) cDNA (MND-MFG-hFVIIIdeltaB). This vector contains multiple modifications in the cis-acting elements within the MoMLV long-terminal repeats (LTR) that prevent the binding of repressive transcription factors. These modifications were previously shown to increase and prolong gene expression in embryonic stem (ES) cells and HSCs. Transduction of BM mesenchymal cells with the MND-MFG-hFVIIIdeltaB retroviral vector resulted in high levels of functional human FVIII in vitro, ranging between 300 +/- 50 SD and 700 +/- 100 SD mU per 106 cells per 24 h. Following xenografting of the transduced human BM cells into immunodeficient NOD-SCID mice, therapeutic hFVIII levels of 12 +/- 10 ng mL-1 were detected in the plasma. Polymerase chain reaction analysis demonstrated long-term engraftment (>3 months) of the human BM mesenchymal cells. The long-term persistence of BM mesenchymal cells in the absence of myelo-ablative conditioning and the therapeutic FVIII levels in vivo underscore the potential usefulness of BM-derived mesenchymal cells for haemophilia gene therapy, as opposed to BM-derived HSCs. Despite the modifications of the MoMLV LTR, FVIII expression declined, which coincided with a decrease in FVIII mRNA transcription levels, indicating that the salutary effect of the LTR modification on transgene expression is not universally applicable to all cell types.