Changes in the accessibility of the HIV-1 Integrase C-terminus in the presence of cellular proteins

<p>Abstract</p> <p>Background</p> <p>Following entry, uncoating, and reverse transcription, a number of cellular proteins become associated with the Human Immunodeficiency Virus type 1 (HIV-1) pre-integration complex (PIC). With the goal of obtaining reagents for the analysis of the HIV-1 PIC composition and localisation, we have constructed functional integrase (IN) and matrix (MA) proteins that can be biotinylated during virus production and captured using streptavidin-coated beads.</p> <p>Results</p> <p>Although the labelled C-terminus allows for the sensitive detection of virion-associated IN, it becomes inaccessible in the presence of cellular proteins. This masking is not dependent on the nature of the tag and does not occur with the tagged MA. It was not observed either with an IN mutant unable to interact with LEDGF/p75, or when LEDGF/p75 was depleted from cells.</p> <p>Conclusion</p> <p>Our observation suggests that a structural rearrangement or oligomerization of the IN protein occurs during the early steps of infection and that this process is related to the presence of LEDGF/p75.</p

Antisense pre-treatment increases gene therapy efficacy in dystrophic muscles

International audienceIn preclinical models for Duchenne muscular dystrophy, dystrophin restoration during adeno-associated virus (AAV)-U7-mediated exon-skipping therapy was shown to decrease drastically after six months in treated muscles. This decline in efficacy is strongly correlated with the loss of the therapeutic AAV genomes, probably due to alterations of the dystrophic myofiber membranes. To improve the membrane integrity of the dystrophic myofibers at the time of AAV-U7 injection, mdx muscles were pre-treated with a single dose of the peptide-phosphorodiamidate morpholino (PPMO) antisense oligonucleotides that induced temporary dystrophin expression at the sarcolemma. The PPMO pre-treatment allowed efficient maintenance of AAV genomes in mdx muscles and enhanced the AAV-U7 therapy effect with a ten-fold increase of the protein level after 6 months. PPMO pre-treatment was also beneficial to AAV-mediated gene therapy with transfer of micro-dystrophin cDNA into muscles. Therefore, avoiding vector genome loss after AAV injection by PPMO pre-treatment would allow efficient long-term restoration of dystrophin and the use of lower and thus safer vector doses for Duchenne patients

Allele‐specific silencing therapy for Dynamin 2‐related dominant centronuclear myopathy

Abstract Rapid advances in allele‐specific silencing by RNA interference established a strategy of choice to cure dominant inherited diseases by targeting mutant alleles. We used this strategy for autosomal‐dominant centronuclear myopathy (CNM), a rare neuromuscular disorder without available treatment due to heterozygous mutations in the DNM2 gene encoding Dynamin 2. Allele‐specific siRNA sequences were developed in order to specifically knock down the human and murine DNM2‐mRNA harbouring the p.R465W mutation without affecting the wild‐type allele. Functional restoration was achieved in muscle from a knock‐in mouse model and in patient‐derived fibroblasts, both expressing the most frequently encountered mutation in patients. Restoring either muscle force in a CNM mouse model or DNM2 function in patient‐derived cells is an essential breakthrough towards future gene‐based therapy for dominant centronuclear myopathy

Splicing efficiency of minor introns in a mouse model of SMA predominantly depends on their branchpoint sequence and can involve the contribution of major spliceosome components

International audienceSpinal muscular atrophy (SMA) is a devastating neurodegenerative disease caused by reduced amounts of the ubiquitously expressed Survival of Motor Neuron (SMN) protein. In agreement with its crucial role in the biogenesis of spliceosomal snRNPs, SMN-deficiency is correlated to numerous splicing alterations in patient cells and various tissues of SMA mouse models. Among the snRNPs whose assembly is impacted by SMN-deficiency, those involved in the minor spliceosome are particularly affected. Importantly, splicing of several, but not all U12-dependent introns has been shown to be affected in different SMA models. Here, we have investigated the molecular determinants of this differential splicing in spinal cords from SMA mice. We show that the branchpoint sequence (BPS) is a key element controlling splicing efficiency of minor introns. Unexpectedly, splicing of several minor introns with suboptimal BPS is not affected in SMA mice. Using in vitro splicing experiments and oligonucleotides targeting minor or major snRNAs, we show for the first time that splicing of these introns involves both the minor and major machineries. Our results strongly suggest that splicing of a subset of minor introns is not affected in SMA mice because components of the major spliceosome compensate for the loss of minor splicing activity

RFX1 and RFX3 Transcription Factors Interact with the D Sequence of Adeno-Associated Virus Inverted Terminal Repeat and Regulate AAV Transduction

Abstract Adeno-associated virus (AAV) transduction efficiency depends on the way in which cellular proteins process viral genomes in the nucleus. In this study, we have investigated the binding of nuclear proteins to the double stranded D (dsD) sequence of the AAV inverted terminal repeat (ITRs) by electromobility shift assay. We present here several lines of evidence that transcription factors belonging to the RFX protein family bind specifically and selectively to AAV2 and AAV1 dsD sequences. Using supershift experiments, we characterize complexes containing RFX1 homodimers and RFX1/RFX3 heterodimers. Following transduction of HEK-293 cells, the AAV genome can be pulled-down by RFX1 and RFX3 antibodies. Moreover, our data suggest that RFX proteins which interact with transcriptional enhancers of several mammalian DNA viruses, can act as regulators of AAV mediated transgene expression

A key aspect to consider for vaccinal protection is the induction of a local line of defense consisting of nonrecirculating tissue-resident memory T cells (TRM), in parallel to the generation of systemic memory CD8+ T cell responses. The potential to induce TRM has now been demonstrated for a number of pathogens and viral vectors. This potential, however, has never been tested for recombinant adeno-associated virus (rAAV) vectors, which are weakly inflammatory and poor transducer of dendritic cells. Using a model rAAV2/1-based vaccine, we determined that a single intradermal immunization with rAAV2/1 vectors in mice induces fully functional TRM at the local site of immunization. The optimal differentiation of rAAV-induced transgene-specific skin TRM was dependent on local transgene expression and additional CD4+ T cell help. Transgene expression in dendritic cells, however, appeared to be dispensable for the priming of transgene-specific skin TRM, suggesting that this process solely depends on the cross-presentation of transgene products. Overall, this study provides needed information to properly assess rAAV vectors as T cell-inducing vaccine carriers.IMPORTANCE rAAVs display numerous characteristics that could make them extremely attractive as vaccine carriers, including an excellent safety profile in humans and great flexibility regarding serotypes and choice of target tissue. Studies addressing the ability of rAAV to induce protective T cell responses, however, are scarce. Notably, the potential to induce a tissue-resident memory T cell response has never been described for rAAV vectors, strongly limiting further interest for their use as vaccine carriers. Using a model rAAV2/1 vaccine delivered to the skin, our study demonstrated that rAAV vectors can induce bona fide skin resident TRM and provides additional clues regarding the cellular mechanisms underlying this process. These results will help widen the field of rAAV applications.

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Intradermal Immunization with rAAV1 Vector Induces Robust Memory CD8+ T Cell Responses Independently of Transgene Expression in DCs

International audienceRecombinant adeno-associated viral (rAAV) vectors exhibit interesting properties as vaccine carriers for their ability to induce long-lasting antibody responses. However, rAAV-based vaccines have been suggested to trigger functionally impaired long-term memory CD8+ T cell responses, in part due to poor dendritic cell (DC) transduction. Such results, albeit limited to intramuscular immunization, undermined the use of rAAV as vaccine vehicles against intracellular pathogens. We report here that intradermal immunization with a model rAAV2/1-based vaccine drives the development of bona fide long-term memory CD8+ T cell responses. The intradermal route of immunization and the presence of potent major histocompatibility complex (MHC) class II responses showed synergistic effects on the overall quantity and quality of systemic long-term effector memory transgene-specific CD8+ T cells being generated against the transgene. Of key interest, we found that the induction of memory cytotoxic T lymphocytes (CTLs) following intradermal immunization was solely dependent on the cross-presentation of skin-expressed transgene products, which appeared highly enhanced as compared to muscle-expressed transgene products. Overall our results highlight key tissue-specific differences in transgene presentation pathway requirements of importance for the design of rAAV-based T cell-inducing vaccines

Benefits of therapy by dynamin-2-mutant-specific silencing are maintained with time in a mouse model of dominant centronuclear myopathy

International audienceDominant dynamin 2 (DNM2) mutations are responsible for the autosomal dominant centronuclear myopathy (AD-CNM), a rare progressive neuromuscular disorder ranging from severe neonatal to mild adult forms. We previously demonstrated that mutant-specific RNA interference is an efficient therapeutic strategy to rescue the muscle phenotype at the onset of the symptoms in the AD-CNM knockin-Dnm2R465W/+ mouse model. Our objective was to evaluate the long-term benefit of the treatment along with the disease time course. We demonstrate here that the complete rescue of the muscle phenotype is maintained for at least 1 year after a single injection of adeno-associated virus expressing the mutant-specific short hairpin RNA (shRNA). This was achieved by a maintained reduction of the mutant Dnm2 transcript. Moreover, this long-term study uncovers a pathological accumulation of DNM2 protein occurring with age in the mouse model and prevented by the treatment. Conversely, a physiological DNM2 protein decrease with age was observed in muscles from wild-type mice. Therefore, this study highlights a new potential pathophysiological mechanism linked to mutant protein accumulation and underlines the importance of DNM2 protein expression level for proper muscle function. Overall, these results strengthen the allele-specific silencing approach as a robust, safe, and efficient therapy for AD-CNM

Benefits of therapy by Dynamin 2 mutant specific silencing are maintained with time in a mouse model of dominant centronuclear myopathy

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