Death after High-Dose rAAV9 Gene Therapy in a Patient with Duchenne's Muscular Dystrophy.

We treated a 27-year-old patient with Duchenne's muscular dystrophy (DMD) with recombinant adeno-associated virus (rAAV) serotype 9 containing dSaCas9 (i.e., "dead" Staphylococcus aureus Cas9, in which the Cas9 nuclease activity has been inactivated) fused to VP64; this transgene was designed to up-regulate cortical dystrophin as a custom CRISPR-transactivator therapy. The dose of rAAV used was 1×1014 vector genomes per kilogram of body weight. Mild cardiac dysfunction and pericardial effusion developed, followed by acute respiratory distress syndrome (ARDS) and cardiac arrest 6 days after transgene treatment; the patient died 2 days later. A postmortem examination showed severe diffuse alveolar damage. Expression of transgene in the liver was minimal, and there was no evidence of AAV serotype 9 antibodies or effector T-cell reactivity in the organs. These findings indicate that an innate immune reaction caused ARDS in a patient with advanced DMD treated with high-dose rAAV gene therapy. (Funded by Cure Rare Disease.).

Cell-Mediated Immunity to NAGLU Transgene Following Intracerebral Gene Therapy in Children With Mucopolysaccharidosis Type IIIB Syndrome.

Mucopolysaccharidosis type IIIB syndrome (Sanfilippo disease) is a rare autosomic recessif disorder caused by mutations in the α-N-acetylglucosaminidase (NAGLU) gene coding for a lysosomal enzyme, leading to neurodegeneration and progressive deterioration of cognitive abilities in affected children. To supply the missing enzyme, several recent human gene therapy trials relied on the deposit of adeno-associated virus (AAV) vectors directly into the brain. We reported safety and efficacy of an intracerebral therapy in a phase 1/2 clinical trial (https://clinicaltrials.gov/ct2/show/NCT03300453), with a recombinant AAV serotype 2/5 (rAAV2/5) coding human NAGLU in four children with MPS IIIB syndrome receiving immunosuppression. It was reported that AAV-mediated gene therapies might elicit a strong host immune response resulting in decreased transgene expression. To address this issue, we performed a comprehensive analysis of cellular immunity and cytokine patterns generated against the therapeutic enzyme in the four treated children over 5.5 years of follow-up. We report the emergence of memory and polyfunctional CD4+ and CD8+ T lymphocytes sensitized to the transgene soon after the start of therapy, and appearing in peripheral blood in waves throughout the follow-up. However, this response had no apparent impact on CNS transgene expression, which remained stable 66 months after surgery, possibly a consequence of the long-term immunosuppressive treatment. We also report that gene therapy did not trigger neuroinflammation, evaluated through the expression of cytokines and chemokines in patients' CSF. Milder disease progression in the youngest patient was found associated with low level and less differentiated circulating NAGLU-specific T cells, together with the lack of proinflammatory cytokines in the CSF. Findings in this study support a systematic and comprehensive immunomonitoring approach for understanding the impact immune reactions might have on treatment safety and efficacy of gene therapies.

ONCR-177, an Oncolytic HSV-1 Designed to Potently Activate Systemic Antitumor Immunity.

ONCR-177 is an engineered recombinant oncolytic herpes simplex virus (HSV) with complementary safety mechanisms, including tissue-specific miRNA attenuation and mutant UL37 to inhibit replication, neuropathic activity, and latency in normal cells. ONCR-177 is armed with five transgenes for IL12, FLT3LG (extracellular domain), CCL4, and antagonists to immune checkpoints PD-1 and CTLA-4. In vitro assays demonstrated that targeted miRNAs could efficiently suppress ONCR-177 replication and transgene expression, as could the HSV-1 standard-of-care therapy acyclovir. Although ONCR-177 was oncolytic across a panel of human cancer cell lines, including in the presence of type I IFN, replication was suppressed in human pluripotent stem cell-derived neurons, cardiomyocytes, and hepatocytes. Dendritic cells activated with ONCR-177 tumor lysates efficiently stimulated tumor antigen-specific CD8+ T-cell responses. In vivo, biodistribution analyses suggested that viral copy number and transgene expression peaked approximately 24 to 72 hours after injection and remained primarily within the injected tumor. Intratumoral administration of ONCR-177 mouse surrogate virus, mONCR-171, was efficacious across a panel of syngeneic bilateral mouse tumor models, resulting in partial or complete tumor regressions that translated into significant survival benefits and to the elicitation of a protective memory response. Antitumor effects correlated with local and distant intratumoral infiltration of several immune effector cell types, consistent with the proposed functions of the transgenes. The addition of systemic anti-PD-1 augmented the efficacy of mONCR-171, particularly for abscopal tumors. Based in part upon these preclinical results, ONCR-177 is being evaluated in patients with metastatic cancer (ONCR-177-101, NCT04348916).

Evading the AAV Immune Response in Mucopolysaccharidoses.

The humoral immune response elicited by adeno-associated virus (AAV)-mediated gene therapy for the treatment of mucopolysaccharidoses (MPS) poses a significant challenge to achieving therapeutic levels of transgene expression. Antibodies targeting the AAV capsid as well as the transgene product diminish the production of glycosaminoglycan (GAG)-degrading enzymes essential for the treatment of MPS. Patients who have antibodies against AAV capsid increase in number with age, serotype, and racial background and are excluded from the clinical trials at present. In addition, patients who have undergone AAV gene therapy are often excluded from the additional AAV gene therapy with the same serotype, since their acquired immune response (antibody) against AAV will limit further efficacy of treatment. Several methods are being developed to overcome this immune response, such as novel serotype design, antibody reduction by plasmapheresis and immunosuppression, and antibody evasion using empty capsids and enveloped AAV vectors. In this review, we examine the mechanisms of the anti-AAV humoral immune response and evaluate the strengths and weaknesses of current evasion strategies in order to provide an evidence-based recommendation on evading the immune response for future AAV-mediated gene therapies for MPS.

Simian adenovirus vector production for early-phase clinical trials: A simple method applicable to multiple serotypes and using entirely disposable product-contact components.

A variety of Good Manufacturing Practice (GMP) compliant processes have been reported for production of non-replicating adenovirus vectors, but important challenges remain. Most clinical development of adenovirus vectors now uses simian adenoviruses or rare human serotypes, whereas reported manufacturing processes mainly use serotypes such as AdHu5 which are of questionable relevance for clinical vaccine development. Many clinically relevant vaccine transgenes interfere with adenovirus replication, whereas most reported process development uses selected antigens or even model transgenes such as fluorescent proteins which cause little such interference. Processes are typically developed for a single adenovirus serotype - transgene combination, requiring extensive further optimization for each new vaccine. There is a need for rapid production platforms for small GMP batches of non-replicating adenovirus vectors for early-phase vaccine trials, particularly in preparation for response to emerging pathogen outbreaks. Such platforms must be robust to variation in the transgene, and ideally also capable of producing adenoviruses of more than one serotype. It is also highly desirable for such processes to be readily implemented in new facilities using commercially available single-use materials, avoiding the need for development of bespoke tools or cleaning validation, and for them to be readily scalable for later-stage studies. Here we report the development of such a process, using single-use stirred-tank bioreactors, a transgene-repressing HEK293 cell - promoter combination, and fully single-use filtration and ion exchange components. We demonstrate applicability of the process to candidate vaccines against rabies, malaria and Rift Valley fever, each based on a different adenovirus serotype. We compare performance of a range of commercially available ion exchange media, including what we believe to be the first published use of a novel media for adenovirus purification (NatriFlo® HD-Q, Merck). We demonstrate the need for minimal process individualization for each vaccine, and that the product fulfils regulatory quality expectations. Cell-specific yields are at the upper end of those previously reported in the literature, and volumetric yields are in the range 1 × 1013 - 5 × 1013 purified virus particles per litre of culture, such that a 2-4 L process is comfortably adequate to produce vaccine for early-phase trials. The process is readily transferable to any GMP facility with the capability for mammalian cell culture and aseptic filling of sterile products.

Targeting a Pre-existing Anti-transgene T Cell Response for Effective Gene Therapy of MPS-I in the Mouse Model of the Disease.

Mucopolysaccharidosis type I (MPS-I) is a severe genetic disease caused by a deficiency of the alpha-L-iduronidase (IDUA) enzyme. Ex vivo hematopoietic stem cell (HSC) gene therapy is a promising therapeutic approach for MPS-I, as demonstrated by preclinical studies performed in naive MPS-I mice. However, after enzyme replacement therapy (ERT), several MPS-I patients develop anti-IDUA immunity that may jeopardize ex vivo gene therapy efficacy. Here we treat MPS-I mice with an artificial immunization protocol to mimic the ERT effect in patients, and we demonstrate that IDUA-corrected HSC engraftment is impaired in pre-immunized animals by IDUA-specific CD8+ T cells spared by pre-transplant irradiation. Conversely, humoral anti-IDUA immunity does not impact on IDUA-corrected HSC engraftment. The inclusion of lympho-depleting agents in pre-transplant conditioning of pre-immunized hosts allowes rescue of IDUA-corrected HSC engraftment, which is proportional to CD8+ T cell eradication. Overall, these data demonstrate the relevance of pre-existing anti-transgene T cell immunity on ex vivo HSC gene therapy, and they suggest the application of tailored immune-depleting treatments, as well as a deeper immunological characterization of patients, to safeguard the therapeutic effects of ex vivo HSC gene therapy in immunocompetent hosts.

A pathway-focused RT-qPCR array study on immune relevant genes in rainbow trout (Oncorhynchus mykiss) harboring cecropin P1 transgene.

Recently, our laboratory had produced five families of transgenic rainbow trout harboring cecropin P1 transgene, and via repeated challenge studies these fish exhibited a significant elevation of resistance to infection by microbial pathogens. By cDNA microarray and mRNA deep sequencing (mRNA-seq) analyses on two of the five families of cecropin P1 transgenic fish, differentially expressed genes (DEGs) relevant to the innate and adaptive immune pathways in three different immune-related tissues, (i.e. spleen, kidney and liver) were profiled. These results supported our hypothesis that in addition to its direct microbicidal activity, the transgene product of cecropin P1 induces immunomodulatory activity in the transgenic host. Here, we have adapted the technique of quantitative reverse transcription real time PCR (RT-qPCR) array to analyze the expression of genes relevant to the innate and adaptive immune pathways in the rest three families. A RT-qPCR array was constructed with oligonucleotide primers of fifty-two innate/adaptive immune relevant DEGs shown to be the most perturbed by cecropin P1 transgene product in previous studies. Messenger RNA isolated from the spleen, kidney and liver of transgenic fish and non-transgenic fish control were studied on this array. Results of RT-qPCR array revealed that statistically significant perturbations of gene expression were detected in pathways of cytokine/chemokine signaling, Toll-like receptor signaling, complement cascade, antigen processing/presentation, lysosomal phagocytosis and leukocyte trans-endothelial migration in the transgenic spleen; extracellular matrix (ECM) organization and leukocyte trans-endothelial migration pathways in the transgenic kidney; lysosomal activity pathway in the transgenic liver. Furthermore, genes related to the pathways of the peroxisome proliferator-activated receptors (PPAR) signaling, lipid metabolism process and arachidonic acid metabolism were also impacted in the transgenic liver. Findings of the current study are in good agreement with those discoveries in previous two transgenic families by cDNA microarray and mRNA-seq analyses.

Liver induced transgene tolerance with AAV vectors.

Immune tolerance is a vital component of immunity, as persistent activation of immune cells causes significant tissue damage and loss of tolerance leads to autoimmunity. Likewise, unwanted immune responses can occur in inherited disorders, such as hemophilia and Pompe disease, in which patients lack any expression of protein, during treatment with enzyme replacement therapy, or gene therapy. While the liver has long been known as being tolerogenic, it was only recently appreciated in the last decade that liver directed adeno-associated virus (AAV) gene therapy can induce systemic tolerance to a transgene. In this review, we look at the mechanisms behind liver induced tolerance, discuss different factors influencing successful tolerance induction with AAV, and applications where AAV mediated tolerance may be helpful.

Cross-Presentation of Skin-Targeted Recombinant Adeno-associated Virus 2/1 Transgene Induces Potent Resident Memory CD8+ T Cell Responses.

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.

Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8+ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector.

Upon viral infection, antigen-specific CD8+ cytotoxic T cells (CTLs) arise and contribute to the elimination of infected cells to prevent the spread of pathogens. Therefore, the frequency of antigen-specific CTLs is indicative of the strength of the T cell response against a specific antigen. Such analysis is important in basic immunology, vaccine development, cancer immunobiology and the adaptive immunology. In the vaccine field, the CTL response directed against components of a viral vector co-determines how effective the generation of antigen-specific cells against the antigen of interest (i.e., transgene) is. Antigen-specific CTLs can either be detected by stimulation with specific peptides followed by intracellular cytokine staining or by the direct staining of antigen-specific T cell receptors (TCRs) and analysis by flow cytometry. The first method is rather time-consuming since it requires sacrificing of animals to isolate cells from organs. Also, it requires isolation of blood from small animals which is difficult to perform. The latter method is rather fast, can be easily done with small amounts of blood and is not dependent on specific effector functions, such as cytolytic activity. MHC tetramers are an ideal tool to detect antigen-specific TCRs. Here, we describe a protocol to simultaneously detect antigen-specific CTLs for the immunodominant peptides of the viral vector VSV-GP (LCMV-GP, VSV-NP) and transgenes (OVA, HPV 16 E7, eGFP) by MHC I tetramer staining and flow cytometry. Staining is possible either directly from blood or from single cell suspensions of organs, such as spleen. Blood or single cell suspensions of organs are incubated with tetramers. After staining with antibodies against CD3 and CD8, antigen-specific CTLs are quantified by flow cytometry. Optionally, antibodies against CD43, CD44, CD62L or others can be included to determine the activation status of antigen-specific CD8+T cells and to discriminate between naïve and effector cells.

Sustained AAV9-mediated expression of a non-self protein in the CNS of non-human primates after immunomodulation.

A critical issue in transgene delivery studies is immune reactivity to the transgene- encoded protein and its impact on sustained gene expression. Here, we test the hypothesis that immunomodulation by rapamycin can decrease immune reactivity after intrathecal AAV9 delivery of a transgene (GFP) in non-human primates, resulting in sustained GFP expression in the CNS. We show that rapamycin treatment clearly reduced the overall immunogenicity of the AAV9/GFP vector by lowering GFP- and AAV9-specific antibody responses, and decreasing T cell responses including cytokine and cytolytic effector responses. Spinal cord GFP protein expression was sustained for twelve weeks, with no toxicity. Immune correlates of robust transgene expression include negligible GFP-specific CD4 and CD8 T cell responses, absence of GFP-specific IFN-γ producing T cells, and absence of GFP-specific cytotoxic T cells, which support the hypothesis that decreased T cell reactivity results in sustained transgene expression. These data strongly support the use of modest doses of rapamycin to modulate immune responses for intrathecal gene therapies, and potentially a much wider range of viral vector-based therapeutics.

Optimization of human papillomavirus (HPV) type 16 E7-expressing lactobacillus-based vaccine for induction of mucosal E7-specific IFNγ-producing cells.

Therapeutic HPV vaccine is an agent to induce E7-specific Th1 immune responses to treat cervical neoplasia (CIN2-3). Our previous clinical trial has demonstrated that oral administration of HPV16 E7-expressing Lactobacillus casei (L. casei), GLBL101c, resulted in the regression of HPV16-related CIN3. Here we examined optimization of the E7-expressing L. casei for induction of the mucosal immune responses to E7. Various doses of HPV16 E7 molecule were displayed on the L. casei. Immunization with E7-bound L. casei showed the induction of E7-specific mucosal IFNγ-producing cells was dependent on displayed E7-doses but saturated beyond 0.3 µg/108 cells. A new agent, L. casei with endogenous expression of E7 (IGMKK16E7), showed the optimal amount of displayed-E7. Immunization with IGMKK16E7 demonstrated 4-fold higher induction of E7-specific mucosal IFNγ-producing cells when compared with the former one. Our new system provided the optimal E7-expressing L. casei for displayed-E7 amount and induction of mucosal Th1 immune response.

Gene Therapy With Regulatory T Cells: A Beneficial Alliance.

Gene therapy aims to replace a defective or a deficient protein at therapeutic or curative levels. Improved vector designs have enhanced safety, efficacy, and delivery, with potential for lasting treatment. However, innate and adaptive immune responses to the viral vector and transgene product remain obstacles to the establishment of therapeutic efficacy. It is widely accepted that endogenous regulatory T cells (Tregs) are critical for tolerance induction to the transgene product and in some cases the viral vector. There are two basic strategies to harness the suppressive ability of Tregs: in vivo induction of adaptive Tregs specific to the introduced gene product and concurrent administration of autologous, ex vivo expanded Tregs. The latter may be polyclonal or engineered to direct specificity to the therapeutic antigen. Recent clinical trials have advanced adoptive immunotherapy with Tregs for the treatment of autoimmune disease and in patients receiving cell transplants. Here, we highlight the potential benefit of combining gene therapy with Treg adoptive transfer to achieve a sustained transgene expression. Furthermore, techniques to engineer antigen-specific Treg cell populations, either through reprogramming conventional CD4+ T cells or transferring T cell receptors with known specificity into polyclonal Tregs, are promising in preclinical studies. Thus, based upon these observations and the successful use of chimeric (IgG-based) antigen receptors (CARs) in antigen-specific effector T cells, different types of CAR-Tregs could be added to the repertoire of inhibitory modalities to suppress immune responses to therapeutic cargos of gene therapy vectors. The diverse approaches to harness the ability of Tregs to suppress unwanted immune responses to gene therapy and their perspectives are reviewed in this article.

Firewalls Prevent Systemic Dissemination of Vectors Derived from Human Adenovirus Type 5 and Suppress Production of Transgene-Encoded Antigen in a Murine Model of Oral Vaccination.

To define the bottlenecks that restrict antigen expression after oral administration of viral-vectored vaccines, we tracked vectors derived from the human adenovirus type 5 at whole body, tissue, and cellular scales throughout the digestive tract in a murine model of oral delivery. After intragastric administration of vectors encoding firefly luciferase or a model antigen, detectable levels of transgene-encoded protein or mRNA were confined to the intestine, and restricted to delimited anatomical zones. Expression of luciferase in the form of multiple small bioluminescent foci in the distal ileum, cecum, and proximal colon suggested multiple crossing points. Many foci were unassociated with visible Peyer's patches, implying that transduced cells lay in proximity to villous rather than follicle-associated epithelium, as supported by detection of transgene-encoded antigen in villous epithelial cells. Transgene-encoded mRNA but not protein was readily detected in Peyer's patches, suggesting that post-transcriptional regulation of viral gene expression might limit expression of transgene-encoded antigen in this tissue. To characterize the pathways by which the vector crossed the intestinal epithelium and encountered sentinel cells, a fluorescent-labeled vector was administered to mice by the intragastric route or inoculated into ligated intestinal loops comprising a Peyer's patch. The vector adhered selectively to microfold cells in the follicle-associated epithelium, and, after translocation to the subepithelial dome region, was captured by phagocytes that expressed CD11c and lysozyme. In conclusion, although a large number of crossing events took place throughout the intestine within and without Peyer's patches, multiple firewalls prevented systemic dissemination of vector and suppressed production of transgene-encoded protein in Peyer's patches.

Adenoviral Type 35 and 26 Vectors with a Bidirectional Expression Cassette in the E1 Region Show an Improved Genetic Stability Profile and Potent Transgene-Specific Immune Response.

Genetic vaccines based on replication-incompetent adenoviral (AdV) vectors are currently in clinical development. Monovalent AdV vectors express one antigen from an expression cassette placed in most cases in the E1 region. For many vaccines, inclusion of several antigens is necessary in order to raise protective immunity and/or target more than one pathogen or pathogen strain. On the basis of the current technology, a mix of several monovalent vectors can be employed. However, a mix of the standard monovalent AdV vectors may not be optimal with respect to manufacturing costs and the final dose per vector in humans. Alternatively, a variety of bivalent recombinant AdV vector approaches is described in the literature. It remains unclear whether all strategies are equally suitable for clinical development while preserving all the beneficial properties of the monovalent AdV (e.g., immunogenic potency). Therefore, a thorough assessment of different bivalent AdV strategies was performed in a head-to-head fashion compared with the monovalent benchmark. The vectors were tested for rescue efficiency, genetic stability, transgene expression, and potency to induce transgene-specific immune responses. We report that the vector expressing multiple antigens from a bidirectional expression cassette in E1 shows a better genetic stability profile and a potent transgene-specific immune response compared with the other tested bivalent vectors.

New gorilla adenovirus vaccine vectors induce potent immune responses and protection in a mouse malaria model.

BACKGROUND: A DNA-human Ad5 (HuAd5) prime-boost malaria vaccine has been shown to protect volunteers against a controlled human malaria infection. The potency of this vaccine, however, appeared to be affected by the presence of pre-existing immunity against the HuAd5 vector. Since HuAd5 seroprevalence is very high in malaria-endemic areas of the world, HuAd5 may not be the most appropriate malaria vaccine vector. This report describes the evaluation of the seroprevalence, immunogenicity and efficacy of three newly identified gorilla adenoviruses, GC44, GC45 and GC46, as potential malaria vaccine vectors. RESULTS: The seroprevalence of GC44, GC45 and GC46 is very low, and the three vectors are not efficiently neutralized by human sera from Kenya and Ghana, two countries where malaria is endemic. In mice, a single administration of GC44, GC45 and GC46 vectors expressing a murine malaria gene, Plasmodium yoelii circumsporozoite protein (PyCSP), induced robust PyCSP-specific T cell and antibody responses that were at least as high as a comparable HuAd5-PyCSP vector. Efficacy studies in a murine malaria model indicated that a prime-boost regimen with DNA-PyCSP and GC-PyCSP vectors can protect mice against a malaria challenge. Moreover, these studies indicated that a DNA-GC46-PyCSP vaccine regimen was significantly more efficacious than a DNA-HuAd5-PyCSP regimen. CONCLUSION: These data suggest that these gorilla-based adenovectors have key performance characteristics for an effective malaria vaccine. The superior performance of GC46 over HuAd5 highlights its potential for clinical development.

5T4 oncofoetal glycoprotein: an old target for a novel prostate cancer immunotherapy.

The tumour-associated antigen 5T4 is an attractive target for cancer immunotherapy. However to date, reported 5T4-specific cellular immune responses induced by various immunisation platforms have been largely weak or non-existent. In the present study, we have evaluated a heterologous prime boost regime based on the simian adenovirus ChAdOx1 and modified vaccinia virus Ankara (MVA) expressing 5T4 for immunogenicity and tumour protective efficacy in a mouse cancer model. Vaccination-induced immune responses were strong, durable and attributable primarily to CD8+ T cells. By comparison, homologous MVA vaccination regimen did not induce detectable 5T4-specific T cell responses. ChAdOx1-MVA vaccinated mice were completely protected against subsequent B16 melanoma challenge, but in therapeutic settings this regime was only modestly effective in delaying tumour outgrowth. Concomitant delivery of the vaccine with monoclonal antibodies (mAbs) targeting immune checkpoint regulators LAG-3, PD-1 or PD-L1 demonstrated that the combination of vaccine with anti PD-1 mAb could significantly delay tumour growth and increase overall survival of tumour-bearing mice. Our findings support a translation of the combinatorial approach based on the heterologous ChAdOx1-MVA vaccination platform with immune checkpoint blockade into the clinic for the treatment of 5T4-positive tumours such as prostate, renal, colorectal, gastric, ovarian, lung cancer and mesothelioma.

Future Prospects for the Development of Cost-Effective Adenovirus Vaccines.

Vaccination is one of the most efficient tools for disease prevention, and a continuously growing field of research. However, despite progress, we still need more efficient and cost-effective vaccines that would improve access to those in need. In this review, we will describe the status of virus-vectored vaccine technology with a focus on adenoviral-based vaccines. Adenovirus (Ad) vaccines have proven to be efficient in military vaccinations against Ad4 and Ad7 and as highly efficient vectored vaccines against rabies. The question of how other adenovirus-based vaccines can become as efficient as the rabies vaccine is the underlying theme in this review. Here, we will first give an overview of the basic properties of vectored vaccines, followed by an introduction to the characteristics of adenoviral vectors and previously tested modifications of the vector backbone and expression cassettes, with a focus on how they can contribute to increased vaccine cost-effectiveness. Finally, we will highlight a few successful examples of research that have attempted to improve the use of adenoviral-based vaccines by improving the transgene immunogenicity.

Systemic AAV8-Mediated Gene Therapy Drives Whole-Body Correction of Myotubular Myopathy in Dogs.

X-linked myotubular myopathy (XLMTM) results from MTM1 gene mutations and myotubularin deficiency. Most XLMTM patients develop severe muscle weakness leading to respiratory failure and death, typically within 2 years of age. Our objective was to evaluate the efficacy and safety of systemic gene therapy in the p.N155K canine model of XLMTM by performing a dose escalation study. A recombinant adeno-associated virus serotype 8 (rAAV8) vector expressing canine myotubularin (cMTM1) under the muscle-specific desmin promoter (rAAV8-cMTM1) was administered by simple peripheral venous infusion in XLMTM dogs at 10 weeks of age, when signs of the disease are already present. A comprehensive analysis of survival, limb strength, gait, respiratory function, neurological assessment, histology, vector biodistribution, transgene expression, and immune response was performed over a 9-month study period. Results indicate that systemic gene therapy was well tolerated, prolonged lifespan, and corrected the skeletal musculature throughout the body in a dose-dependent manner, defining an efficacious dose in this large-animal model of the disease. These results support the development of gene therapy clinical trials for XLMTM.

Comparison of Serum rAAV Serotype-Specific Antibodies in Patients with Duchenne Muscular Dystrophy, Becker Muscular Dystrophy, Inclusion Body Myositis, or GNE Myopathy.

Recombinant adeno-associated virus (rAAV) is a commonly used gene therapy vector for the delivery of therapeutic transgenes in a variety of human diseases, but pre-existing serum antibodies to viral capsid proteins can greatly inhibit rAAV transduction of tissues. Serum was assayed from patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), inclusion body myositis (IBM), and GNE myopathy (GNE). These were compared to serum from otherwise normal human subjects to determine the extent of pre-existing serum antibodies to rAAVrh74, rAAV1, rAAV2, rAAV6, rAAV8, and rAAV9. In almost all cases, patients with measurable titers to one rAAV serotype showed titers to all other serotypes tested, with average titers to rAAV2 being highest in all instances. Twenty-six percent of all young normal subjects (<18 years old) had measurable rAAV titers to all serotypes tested, and this percentage increased to almost 50% in adult normal subjects (>18 years old). Fifty percent of all IBM and GNE patients also had antibody titers to all rAAV serotypes, while only 18% of DMD and 0% of BMD patients did. In addition, serum-naïve macaques treated systemically with rAAVrh74 could develop cross-reactive antibodies to all other serotypes tested at 24 weeks post treatment. These data demonstrate that most DMD and BMD patients should be amenable to vascular rAAV-mediated treatment without the concern of treatment blockage by pre-existing serum rAAV antibodies, and that serum antibodies to rAAVrh74 are no more common than those for rAAV6, rAAV8, or rAAV9.