Search Results (10)

Recombinant adeno-associated viruses (rAAVs) have become one of the leading gene therapies for treating a variety of diseases. One factor contributing to rAAVs’ success is the fact that a wide variety of tissue types can be transduced by different serotypes. However, one commonality amongst most serotypes is the high propensity for liver transduction when rAAVs are administered peripherally. One of the few exceptions is the naturally occurring clade F AAV hematopoietic stem cell 16 (AAVHSC16). AAVHSC16 represents an interesting capsid in that it shows minimal liver transduction when injected peripherally. For capsids other than AAVHSC16, targeting non-liver tissues via peripheral AAV injection represents a challenge due to the high liver transduction. Thus, there is a demand for liver-de-targeted rAAV vectors. The rational design of rAAV capsids relies on current knowledge to design improved capsids and represents one means of developing capsids with reduced liver transduction. Here, we utilized data from the AAVHSC16 capsid to rationally design four non-clade F rAAV capsids that result in reduced liver transduction following peripheral injection. Full article

Virus-like particles constitute versatile vectors that can be used as vaccine platforms in many fields from infectiology and more recently to oncology. We previously designed non-infectious adenovirus-inspired 60-mer dodecahedric virus-like particles named ADDomers displaying on their surface either a short epitope or a large tumor/viral antigen. In this work, we explored for the first time the immunogenicity of ADDomers exhibiting melanoma-derived tumor antigen/epitope and their impact on the features of human dendritic cell (DC) subsets. We first demonstrated that ADDomers displaying tumor epitope/antigen elicit a strong immune-stimulating potential of human DC subsets (cDC2s, cDC1s, pDCs), which were able to internalize and cross-present tumor antigen, and subsequently cross-prime antigen-specific T-cell responses. To further limit off-target effects and enhance DC targeting, we engineered specific motifs to de-target epithelial cells and improve DCs’ addressing. The improved engineered platform making it possible to display large antigen represents a tool to overcome the barrier of immune allele restriction, broadening the immune response, and paving the way to its potential utilization in humans as an off-the-shelf vaccine. Full article

Carcinomas are characterized by a widespread upregulation of intercellular junctions that create a barrier to immune response and drug therapy. Desmoglein 2 (DSG2) represents such a junction protein and serves as one adenovirus receptor. Importantly, the interaction between human adenovirus type 3 (Ad3) and DSG2 leads to the shedding of the binding domain followed by a decrease in the junction protein expression and transient tight junction opening. Junction opener 4 (JO-4), a small recombinant protein derived from the Ad3 fiber knob, was previously developed with a higher affinity to DSG2. JO-4 protein has been proven to enhance the effects of antibody therapy and chemotherapy and is now considered for clinical trials. However, the effect of the JO4 mutation in the context of a virus remains insufficiently studied. Therefore, we introduced the JO4 mutation to various adenoviral vectors to explore their infection properties. In the current experimental settings and investigated cell lines, the JO4-containing vectors showed no enhanced transduction compared with their parental vectors in DSG2-high cell lines. Moreover, in DSG2-low cell lines, the JO4 vectors presented a rather weakened effect. Interestingly, DSG2-negative cell line MIA PaCa-2 even showed resistance to JO4 vector infection, possibly due to the negative effect of JO4 mutation on the usage of another Ad3 receptor: CD46. Together, our observations suggest that the JO4 vectors may have an advantage to prevent CD46-mediated sequestration, thereby achieving DSG2-specific transduction. Full article

A major limiting factor for systemically delivered gene therapies is the lack of novel tissue specific AAV (Adeno-associated virus) derived vectors. Bispecific antibodies can be used to redirect AAVs to specific target receptors. Here, we demonstrate that the insertion of a short linear epitope “2E3” derived from human proprotein-convertase subtilisin/kexin type 9 (PCSK9) into different surface loops of the VP capsid proteins can be used for AAV de-targeting from its natural receptor(s), combined with a bispecific antibody-mediated retargeting. We chose to target a set of distinct disease relevant membrane proteins—fibroblast activation protein (FAP), which is upregulated on activated fibroblasts within the tumor stroma and in fibrotic tissues, as well as programmed death-ligand 1 (PD-L1), which is strongly upregulated in many cancers. Upon incubation with a bispecific antibody recognizing the 2E3 epitope and FAP or PD-L1, the bispecific antibody/rAAV complex was able to selectively transduce receptor positive cells. In summary, we developed a novel, rationally designed vector retargeting platform that can target AAVs to a new set of cellular receptors in a modular fashion. This versatile platform may serve as a valuable tool to investigate the role of disease relevant cell types and basis for novel gene therapy approaches. Full article

Our laboratory has pursued the generation of cancer-specific oncolytic herpes simplex viruses (oHSVs) which ensure high efficacy while maintaining a high safety profile. Their blueprint included retargeting to a Tumor-Associated Antigen, e.g., HER2, coupled to detargeting from natural receptors to avoid off-target and off-tumor infections and preservation of the full complement of unmodified viral genes. These oHSVs are “fully virulent in their target cancer cells”. The 3rd generation retargeted oHSVs carry two distinct retargeting moieties, which enable infection of a producer cell line and of the target cancer cells, respectively. They can be propagated in an ad hoc Vero cell derivative at about tenfold higher yields than 1st generation recombinants, and more effectively replicate in human cancer cell lines. The R-335 and R-337 prototypes were armed with murine IL-12. Intratumorally-administered R-337 conferred almost complete protection from LLC-1-HER2 primary tumors, unleashed the tumor microenvironment immunosuppression, synergized with the checkpoint blockade and conferred long-term vaccination against distant challenge tumors. In summary, the problem intrinsic to the propagation of retargeted oHSVs—which strictly require cells positive for targeted receptors—was solved in 3rd generation viruses. They are effective as immunotherapeutic agents against primary tumors and as antigen-agnostic vaccines. Full article

Lentiviral-mediated integration of a CFTR transgene cassette into airway basal cells is a strategy being considered for cystic fibrosis (CF) cell-based therapies. However, CFTR expression is highly regulated in differentiated airway cell types and a subset of intermediate basal cells destined to differentiate. Since basal stem cells typically do not express CFTR, suppressing the CFTR expression from the lentiviral vector in airway basal cells may be beneficial for maintaining their proliferative capacity and multipotency. We identified miR-106b as highly expressed in proliferating airway basal cells and extinguished in differentiated columnar cells. Herein, we developed lentiviral vectors with the miR-106b-target sequence (miRT) to both study miR-106b regulation during basal cell differentiation and detarget CFTR expression in basal cells. Given that miR-106b is expressed in the 293T cells used for viral production, obstacles of viral genome integrity and titers were overcome by creating a 293T-B2 cell line that inducibly expresses the RNAi suppressor B2 protein from flock house virus. While miR-106b vectors effectively detargeted reporter gene expression in proliferating basal cells and following differentiation in the air–liquid interface and organoid cultures, the CFTR-miRT vector produced significantly less CFTR-mediated current than the non-miR-targeted CFTR vector following transduction and differentiation of CF basal cells. These findings suggest that miR-106b is expressed in certain airway cell types that contribute to the majority of CFTR anion transport in airway epithelium. Full article

Oncolytic viruses would ideally be of use for systemic therapy to treat disseminated cancer. To do this safely, this may require multiple layers of cancer specificity. The pharmacology and specificity of oncolytic adenoviruses can be modified by (1) physical retargeting, (2) physical detargeting, (3) chemical shielding, or (4) by modifying the ability of viral early gene products to selectively activate in cancer versus normal cells. We explored the utility of these approaches with oncolytic adenovirus serotype 5 (Ad5) in immunocompetent Syrian hamsters bearing subcutaneous HaK tumors. After a single intravenous injection to reach the distant tumors, the physically hepatocyte-detargeted virus Ad5-hexon-BAP was more effective than conditionally replicating Ad5-dl1101/07 with mutations in its E1A protein. When these control or Ad5 treated animals were treated a second time by intratumoral injection, prior exposure to Ad5 did not affect tumor growth, suggesting that anti-Ad immunity neither prevented treatment nor amplified anti-tumor immune responses. Ad5-dl1101/07 was next chemically shielded with polyethylene glycol (PEG). While 5 kDa of PEG blunted pro-inflammatory IL-6 production induced by Ad5-dl1101/07, this shielding reduced Ad oncolytic activity. Full article

Most of the oncolytic herpes simplex viruses (HSVs) exhibit a high safety profile achieved through attenuation. They carry defects in virulence proteins that antagonize host cell response to the virus, including innate response, apoptosis, authophagy, and depend on tumor cell proliferation. They grow robustly in cancer cells, provided that these are deficient in host cell responses, which is often the case. To overcome the attenuation limits, a strategy is to render the virus highly cancer-specific, e.g., by retargeting their tropism to cancer-specific receptors, and detargeting from natural receptors. The target we selected is HER-2, overexpressed in breast, ovarian and other cancers. Entry of wt-HSV requires the essential glycoproteins gD, gH/gL and gB. Here, we reviewed that oncolytic HSV retargeting was achieved through modifications in gD: the addition of a single-chain antibody (scFv) to HER-2 coupled with appropriate deletions to remove part of the natural receptors’ binding sites. Recently, we showed that also gH/gL can be a retargeting tool. The insertion of an scFv to HER-2 at the gH N-terminus, coupled with deletions in gD, led to a recombinant capable to use HER-2 as the sole receptor. The retargeted oncolytic HSVs can be administered systemically by means of carrier cells-forcedly-infected mesenchymal stem cells. Altogether, the retargeted oncolytic HSVs are highly cancer-specific and their replication is not dependent on intrinsic defects of the tumor cells. They might be further modified to express immunomodulatory molecules. Full article

Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans) and “bridging” interactions. “Bridging” interactions refer to coagulation factor binding, namely coagulation factor X (FX), which bridges hepatocyte transduction in vivo through engagement with surface expressed heparan sulfate proteoglycans (HSPGs). These interactions can contribute to the off-target sequestration of Ad5 in the liver and its characteristic dose-limiting hepatotoxicity, thereby significantly limiting the in vivo targeting efficiency and clinical potential of Ad5-based therapeutics. To date, various approaches to retargeting adenoviruses (Ad) have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon), pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with non-human Ad species or with capsid proteins derived from other viral families, hexon hypervariable region (HVR) substitutions and adapter-based conjugation/crosslinking of scFv, growth factors or monoclonal antibodies directed against surface-expressed target antigens. In order to maximize retargeting, strategies which permit detargeting from undesirable interactions between the Ad capsid and components of the circulatory system (e.g. coagulation factors, erythrocytes, pre-existing neutralizing antibodies), can be employed simultaneously. Detargeting can be achieved by genetic ablation of native receptor-binding determinants, ablation of “bridging interactions” such as those which occur between the hexon of Ad5 and coagulation factor X (FX), or alternatively, through the use of polymer-coated “stealth” vectors which avoid these interactions. Simultaneous retargeting and detargeting can be achieved by combining multiple genetic and/or chemical modifications. Full article

Cancer gene therapy consists of numerous approaches where the common denominator is utilization of vectors for achieving therapeutic effect. A particularly potent embodiment of the approach is virotherapy, in which the replication potential of an oncolytic virus is directed towards tumor cells to cause lysis, while normal cells are spared. Importantly, the therapeutic effect of the initial viral load is amplified through viral replication cycles and production of progeny virions. All cancer gene therapy approaches rely on a sufficient level of delivery of the anticancer agent into target cells. Thus,enhancement of delivery to target cells, and reduction of delivery to non-target cells, in an approach called transductional targeting, is attractive. Both genetic and non-genetic retargeting strategies have been utilized. However, in the context of oncolytic viruses, it is beneficial to have the specific modification included in progeny virions and hence genetic modification may be preferable. Serotype chimerism utilizes serotype specific differences in receptor usage, liver tropism and seroprevalence in order to gain enhanced infection of target tissue. This review will focus on serotype chimeric adenoviruses for cancer gene therapy applications. Full article