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.

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.

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.

Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions.

Vascular endothelial growth factor (VEGF) stimulates angiogenesis by activating VEGF receptor-2 (VEGFR-2). The role of its homolog, placental growth factor (PlGF), remains unknown. Both VEGF and PlGF bind to VEGF receptor-1 (VEGFR-1), but it is unknown whether VEGFR-1, which exists as a soluble or a membrane-bound type, is an inert decoy or a signaling receptor for PlGF during angiogenesis. Here, we report that embryonic angiogenesis in mice was not affected by deficiency of PlGF (Pgf-/-). VEGF-B, another ligand of VEGFR-1, did not rescue development in Pgf-/- mice. However, loss of PlGF impaired angiogenesis, plasma extravasation and collateral growth during ischemia, inflammation, wound healing and cancer. Transplantation of wild-type bone marrow rescued the impaired angiogenesis and collateral growth in Pgf-/- mice, indicating that PlGF might have contributed to vessel growth in the adult by mobilizing bone-marrow-derived cells. The synergism between PlGF and VEGF was specific, as PlGF deficiency impaired the response to VEGF, but not to bFGF or histamine. VEGFR-1 was activated by PlGF, given that anti-VEGFR-1 antibodies and a Src-kinase inhibitor blocked the endothelial response to PlGF or VEGF/PlGF. By upregulating PlGF and the signaling subtype of VEGFR-1, endothelial cells amplify their responsiveness to VEGF during the 'angiogenic switch' in many pathological disorders.

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.

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.

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.

Development and analysis of retroviral vectors expressing human factor VIII as a potential gene therapy for hemophilia A.

To develop a potential gene therapy strategy for the treatment of hemophilia A, we constructed several retroviral vectors expressing a B-domain-deleted factor VIII (FVIII) cDNA. We confirmed previous reports that when the FVIII cDNA is inserted into a retroviral vector, the vector mRNA is decreased resulting in significantly (100- to 1,000-fold) lower vector titers. In an attempt to overcome this inhibition we pursued two independent strategies. First, site-directed mutagenesis was employed to change the structure of a putative 1.2-kb FVIII RNA inhibitory sequence (INS). Second, the FVIII gene was transcribed from a retroviral vector containing a 5' intron. Results demonstrated that the intron increased FVIII expression up to 20-fold and viral titer up to 40-fold but conservative mutagenesis of the putative FVIII INS region failed to yield a significant increase in FVIII expression or titer. Using the improved FVIII splicing vector, we transduced a variety of cell types and were able to demonstrate relatively high FVIII expression (10-60 ng of FVIII/10(6) cells/24 hr). These results underscore the usefulness of these transduced cell types for potential in vivo delivery of FVIII.

Bone marrow stromal cells as targets for gene therapy of hemophilia A.

Attempts to develop an ex vivo gene therapy strategy for hemophilia A, using either primary T cells or bone marrow (BM) stem/progenitor cells have been unsuccessful, due to the inability of these cell types to express coagulation factor VIII (FVIII). As an alternative, we evaluated the potential of BM-derived stromal cells which can be readily obtained and expanded in vitro. Human and murine BM stromal cells were transduced with an intron-based Moloney murine leukemia virus (MoMLV) retroviral vector expressing a B-domain-deleted human factor VIII cDNA (designated as MFG-FVIIIdeltaB). Transduction efficiencies were increased 10- to 15-fold by phosphate depletion and centrifugation, which obviated the need for selective enrichment of the transduced BM stromal cells. This resulted in high FVIII expression levels in transduced human (180 +/- 4 ng FVIII/10[6] cells per 24 hr) and mouse (900 +/- 130 ng FVIII/10[6] cells per 24 hr) BM stromal cells. Pseudotyping of the MFG-FVIIIdeltaB retroviral vectors with the gibbon ape leukemia virus envelope (GALV-env) resulted in significantly higher transduction efficiencies (100 +/- 20%) and FVIII expression levels (390 +/- 10 ng FVIII/10[6] cells per 24 hr) in transduced human BM stromal cells than with standard amphotropic vectors. This difference in transduction efficiency correlated with the higher titer of the GALV-env pseudotyped viral vectors and with the higher GALV receptor (GLVR-1) versus amphotropic receptor (GLVR-2) mRNA expression levels in human BM stromal cells. These findings demonstrate the potential of BM stromal cells for gene therapy in general and hemophilia A in particular.

Inhibition of human immunodeficiency virus type-1 by retroviral vectors expressing antisense-TAR.

The human immunodeficiency virus type-1 (HIV-1) Tat activation response (TAR) region is essential for Tat-mediated trans-activation of the HIV-1 long terminal repeat (LTR). The TAR element is present on the 5' and 3' ends of all HIV-1 transcripts and is relatively conserved among different HIV-1 isolates. These properties make it an attractive target for anti-HIV-1 gene therapy strategies. We have constructed a Moloney murine leukemia-based retroviral vector that expresses a chimeric tRNA(iMet)-antisense TAR fusion transcript complementary to the HIV-1 TAR region. The potential of this anti-TAR retroviral vector to inhibit HIV-1 was initially tested by transient transfections with an HIV-1-LTR-Tat expression plasmid into HeLa-CAT cells. Anti-TAR inhibited Tat-mediated HIV-1 LTR-driven CAT reporter gene expression in a dose-dependent fashion. The antisense-TAR vector was then used to transduce the human SupT1 T cell line. Cotransfection of these SupT1 cells with a Tat expression plasmid plus an HIV-1 LTR-CAT reporter plasmid resulted in decreased CAT gene expression in comparison to control transduced SupT1 cells. The antisense-TAR engineered SupT1 cell line was then challenged with HIV-1MN.HIV-1 viral production was inhibited in SupT1 cells transduced with the antisense-TAR retroviral vector. Greater inhibition of HIV-1 was observed with antisense-TAR as compared to antisense-Tat expressing retroviral vector. These observations suggest that antisense-TAR retroviral vectors are potentially useful for clinical anti-HIV-1 gene therapy.

Long-term persistence of human bone marrow stromal cells transduced with factor VIII-retroviral vectors and transient production of therapeutic levels of human factor VIII in nonmyeloablated immunodeficient mice.

The potential of using bone marrow (BM)-derived human stromal cells for ex vivo gene therapy of hemophilia A was evaluated. BM stromal cells were transduced with an intron-based Moloney murine leukemia virus (Mo-MuLV) retroviral vector that contained the B domain-deleted human factor VIII (FVIIIdeltaB) cDNA. This FVIII-retroviral vector was pseudotyped with the gibbon ape leukemia virus envelope (GALV-env) to attain higher transduction efficiencies. Using optimized transduction methods, high in vitro FVIII expression levels of 700 to 2500 mU of FVIII/10(6) cells per 24 hr were achieved without selective enrichment of the transduced BM stromal cells. After xenografting of 1.5-3 x 106 engineered BM stromal cells into the spleen of nonobese diabetic severe combined immunodeficient (NOD-SCID) mice, human plasma FVIII levels rose to 13 +/- 4 ng/ml but declined to basal levels by 3 weeks postinjection because of promoter inactivation. About 10% of these stromal cells engrafted in the spleen and persisted for at least 4 months after transplantation in the absence of myeloablative conditioning. No human BM stromal cells could be detected in other organs. These findings indicate that retroviral vector-mediated gene therapy using engineered BM stromal cells may lead to therapeutic levels of FVIII in vivo and that long-term engraftment of human BM stromal cells was achieved in the absence of myeloablative conditioning and without neo-organs. Hence, BM stromal cells may be useful for gene therapy of hemophilia A, provided prolonged expression can be achieved by using alternative promoters.

Tumorigenicity of mouse T lymphoma cells is controlled by the level of major histocompatibility complex class I H-2Kk antigens.

We have previously found that an increased tumorigenicity and spontaneous metastatic potential of BW5147-derived T lymphoma cells was associated with a decrease in major histocompatibility complex (MHC) class I H-2Kk antigen expression. This suggested that H-2Kk antigens may control the tumorigenic potential of BW T lymphoma cells. Our current experiments aimed to prove this association by specifically altering H-2Kk expression by gene transfection. Transfected cells expressing a high level of H-2Kk antigens were significantly less tumorigenic and metastatic after subcutaneous inoculation. However, there was selection in vivo for cells expressing a reduced level of H-2Kk antigens, which concomitantly led to an increased tumorigenicity. These data further confirmed the strong association between H-2Kk expression and tumorigenicity. We subsequently tested whether the immune system is implicated in this phenomenon by inoculating the H-2Kk transfectants into irradiated, immunocompromised recipients. Our results indicate that the reduced tumorigenicity of the BW H-2Kk transfectants is due to an immune rejection mechanism, mediated by CD8+ immune effector cells, as revealed by in vivo depletion experiments with anti-CD8 antibodies. Hence, we hereby demonstrated that H-2Kk antigens increased the immunogenicity of BW cells, via a CD8-dependent mechanism, which consequently reduced their tumorigenicity.

Viral vector-mediated gene therapy for hemophilia.

Hemophilia A and B are hereditary coagulation disorders that result from functional deficiencies of factor VIII (FVIII) or factor IX (FIX), respectively. Current treatment consists of injections with plasma-derived or recombinant clotting factors. Despite the significant clinical benefits of protein replacement therapies, these do not constitute a cure and patients are still at risk of bleeding. Significant progress has been made recently in the development of gene therapy for hemophilia. This has been primarily due to the technical improvements of existing vector systems and the development of new gene delivery methods. Therapeutic and sometimes physiologic levels of FVIII and FIX could be achieved in FVIII- and FIX-deficient mice and hemophilic dogs using different types of viral vectors. In these preclinical studies, long-term correction of the bleeding disorders and in some cases a permanent cure has been realized. However, complications related to the induction of neutralizing antibodies or viral promoter inactivation often precludes stable phenotypic correction. Several gene therapy phase I clinical trials have been initiated in patients suffering from severe hemophilia A or B. The results from the extensive pre-clinical studies and the preliminary clinical data are encouraging. It is likely that successful gene therapy for hemophilia will become a reality at the beginning of this new millennium, serving as the trailblazer for gene therapy of other diseases.

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.

Detection of antigen in the coelomocytes of the earthworm, Eisenia foetida (Annelida).

Earthworms, Eisenia foetida, are able to respond to antigenic stimulation by the formation of the antigen-binding molecules by coelomocytes--the effector cells of annelids' defence reactions. The ability to react with gold-labelled antigen was detected in agranular coelomocytes by electron microscopy. Furthermore, flow cytometry analysis used for quantitative evaluation of antigen binding showed significant increase of both antigen-binding cells and the amount of antigen bound per cell after stimulation. The antigen binding was inhibited by preincubation of cells with several similar proteins, although the most potent inhibitor was the immunizing antigen.

Analysis of trans-dominant mutants of the HIV type 1 Rev protein for their ability to inhibit Rev function, HIV type 1 replication, and their use as anti-HIV gene therapeutics.

The HIV-1 rev gene product facilitates the transport of singly spliced and unspliced HIV-1 transcripts and is necessary for productive HIV-1 infection. On the basis of the previously described trans-dominant Rev mutant M10, four point mutants and one frameshift mutant of the Rev protein were constructed. The mutants were inserted into retroviral expression vectors and analyzed for their ability to inhibit Rev-mediated gene expression. Transient transfection systems were used to screen these new mutants, and each was shown to inhibit expression of a Rev-dependent CAT reporter plasmid. Inhibition of HIV-1 envelope gene expression was tested in the HeLa-T4 cell line and was also shown to be inhibited by the trans-dominant Rev mutants. Retroviral vector producer cell lines were constructed and used to transduce Rev trans-dominant genes into the human T-cell line SupT1. The engineered SupT1 cell lines were then challenged with HIV-1 IIIB and HIV-1 expression was monitored by Northern blot analysis and in situ hybridization. SupT1 cells expressing either a Rev point mutant or the frameshift mutant showed greatly reduced HIV-1 mRNA accumulation and the Rev-dependent singly spliced and unspliced HIV-1 mRNAs were reduced. The kinetics of viral replication following challenge of Rev trans-dominant-engineered SupT1 cells with both HIV-1 IIIB and MN strains was significantly reduced and cells were protected from viral lysis. Viruses that emerge late in infection from Rev trans-dominant-engineered cultures are not resistant to Rev-mediated inhibition. Last, trans-dominant Rev-mediated protection of human CD4+ lymphocytes from challenge with primary HIV-1 patient isolates confirms the potential utility of this system as an anti-HIV-1 gene therapy approach.

[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.