Therapeutic Gene Transfer Research Articles

Chitosan: An overview of biological activities, derivatives, properties, and current advancements in biomedical applications

The second and most often utilized natural polymer is chitosan (CS), a naturally existing amino polysaccharide that is produced by deacetylating chitin. Numerous applications have been the subject of in-depth investigation due to its non-hazardous, biologically compatible, and biodegradable qualities. Chitosan's characteristics, such as mucoadhesion, improved permeability, controlled release of drugs, in situ gelation process, and antibacterial activity, depend on its amino (-NH2) and hydroxyl groups (-OH). This study examines the latest findings in chitosan research, including its characteristics, derivatives, preliminary research, toxic effects, pharmaceutical kinetics and chitosan nanoparticles (CS-NPs) based for non-parenteral delivery of drugs. Chitosan and its derivatives have a wide range of physical and chemical properties that make them highly promising for use in the medicinal and pharmaceutical industries. The characteristics and biological activities of chitosan and its derivative-based nanomaterials for the delivery of drugs, therapeutic gene transfer, delivery of vaccine, engineering tissues, evaluations, and other applications in medicine are highlighted in detail in the current review. Together with the techniques for binding medications to nanoparticles, the application of the nanoparticles was also dictated by their physical properties that were classified and specified. The most recent research investigations on delivery of drugs chitosan nanoparticle-based medication delivery methods applied topically, through the skin, and through the eyes were considered.

Adeno-associated virus perfusion enhanced expression: A commercially scalable, high titer, high quality producer cell line process

With safety and efficacy demonstrated over hundreds of clinical trials in the last 30 years, along with at least six recent global marketing authorizations achieved since 2017, recombinant adeno-associated viruses (rAAVs) have been established asthe leading therapeutic gene transfer vector for rare, monogenic diseases. Significant advances in manufacturing technology have been made in the last few decades to address challenges with GMP production of rAAV products, although yield, cost, scalability, and quality remain a challenge. With transient transfection processes established as a manufacturing platform for multiple commercial AAV products, there remains significant yield, cost, robustness, and scalability constraints that need to be resolved to enable a reliable supply of rAAV products for global patient access. The development of stable producer cell lines for rAAV products has enabled scalability and, in some cases, improvements in productivity. Herein we describe a novel AAV perfusion-enhanced expression (APEX) process, resulting in higher maximum cell densities in the production bioreactor with a 3- to 6-fold increase in volumetric productivity. This process has been successfully demonstrated across multiple serotypes in large scale cell culture with titers approaching 1×1012 GC/mL. The APEX production platform marks a significant leap forward in the efficient and effective manufacturing of rAAV vector products.

Adeno-associated virus vectors for retinal gene therapy in basic research and clinical studies.

Retinal degenerative diseases, including glaucoma, age-related macular degeneration, diabetic retinopathy, and a broad range of inherited retinal diseases, are leading causes of irreversible vision loss and blindness. Gene therapy is a promising and fast-growing strategy to treat both monogenic and multifactorial retinal disorders. Vectors for gene delivery are crucial for efficient and specific transfer of therapeutic gene(s) into target cells. AAV vectors are ideal for retinal gene therapy due to their inherent advantages in safety, gene expression stability, and amenability for directional engineering. The eye is a highly compartmentalized organ composed of multiple disease-related cell types. To determine a suitable AAV vector for a specific cell type, the route of administration and choice of AAV variant must be considered together. Here, we provide a brief overview of AAV vectors for gene transfer into important ocular cell types, including retinal pigment epithelium cells, photoreceptors, retinal ganglion cells, Müller glial cells, ciliary epithelial cells, trabecular meshwork cells, vascular endothelial cells, and pericytes, via distinct injection methods. By listing suitable AAV vectors in basic research and (pre)clinical studies, we aim to highlight the progress and unmet needs of AAV vectors in retinal gene therapy.

Immunogenicity of Recombinant Adeno-Associated Virus (AAV) Vectors for Gene Transfer.

Recombinant adeno-associated viruses (AAVs) have emerged as promising gene delivery vehicles resulting in three US Food and Drug Administration (FDA) and one European Medicines Agency (EMA)-approved AAV-based gene therapies. Despite being a leading platform for therapeutic gene transfer in several clinical trials, host immune responses against the AAV vector and transgene have hampered their widespread application. Multiple factors, including vector design, dose, and route of administration, contribute to the overall immunogenicity of AAVs. The immune responses against the AAV capsid and transgene involve an initial innate sensing. The innate immune response subsequently triggers an adaptive immune response to elicit a robust and specific response against the AAV vector. AAV gene therapy clinical trials and preclinical studies provide important information about the immune-mediated toxicities associated with AAV, yet studies suggest preclinical models fail to precisely predict the outcome of gene delivery in humans. This review discusses the contribution of the innate and adaptive immune response against AAVs, highlighting the challenges and potential strategies to mitigate these responses, thereby enhancing the therapeutic potential of AAV gene therapy.

Cross-species evolution of a highly potent AAV variant for therapeutic gene transfer and genome editing

Recombinant adeno-associated viral (AAV) vectors are a promising gene delivery platform, but ongoing clinical trials continue to highlight a relatively narrow therapeutic window. Effective clinical translation is confounded, at least in part, by differences in AAV biology across animal species. Here, we tackle this challenge by sequentially evolving AAV capsid libraries in mice, pigs and macaques. We discover a highly potent, cross-species compatible variant (AAV.cc47) that shows improved attributes benchmarked against AAV serotype 9 as evidenced by robust reporter and therapeutic gene expression, Cre recombination and CRISPR genome editing in normal and diseased mouse models. Enhanced transduction efficiency of AAV.cc47 vectors is further corroborated in macaques and pigs, providing a strong rationale for potential clinical translation into human gene therapies. We envision that ccAAV vectors may not only improve predictive modeling in preclinical studies, but also clinical translatability by broadening the therapeutic window of AAV based gene therapies.

Lentiviral interferon: A novel method for gene therapy in bladder cancer

Interferon alpha (IFNα) gene therapy is emerging as a new treatment option for patients with non-muscle invasive bladder cancer (NMIBC). Adenoviral vectors expressing IFNα have shown clinical efficacy treating bacillus Calmette-Guerin (BCG)-unresponsive bladder cancer (BLCA). However, transient transgene expression and adenoviral immunogenicity may limit therapeutic activity. Lentiviral vectors can achieve stable transgene expression and are less immunogenic. In this study, we evaluated lentiviral vectors expressing murine IFNα (LV-IFNα) and demonstrate IFNα expression by transduced murine BLCA cell lines, bladder urothelium, and within the urine following intravesical instillation. Murine BLCA cell lines (MB49 and UPPL1541) were sensitive to IFN-mediated cell death after LV-IFNα, whereas BBN975 was inherently resistant. Upregulation of interleukin-6 (IL-6) predicted sensitivity to IFN-mediated cell death mediated by caspase signaling, which when inhibited abrogated IFN-mediated cell killing. Intravesical therapy with LV-IFNα/Syn3 in a syngeneic BLCA model significantly improved survival, and molecular analysis of treated tumors revealed upregulation of apoptotic and immune-cell-mediated death pathways. In particular, biomarker discovery analysis identified three clinically actionable targets, PD-L1, epidermal growth factor receptor (EGFR), and ALDHA1A, in murine tumors treated with LV-IFNα/Syn3. Our findings warrant the comparison of adenoviral and LV-IFNα and the study of novel combination strategies with IFNα gene therapy for the BLCA treatment.

Application of in-vitro-cultured primary hepatocytes to evaluate species translatability and AAV transduction mechanisms of action

Recombinant adeno-associated virus (AAV) is an effective platform for therapeutic gene transfer; however, tissue-tropism differences between species are a challenge for successful translation of preclinical results to humans. We evaluated the use of in vitro primary hepatocyte cultures to predict in vivo liver-directed AAV expression in different species. We assessed whether in vitro AAV transduction assays in cultured primary hepatocytes from mice, nonhuman primates (NHPs), and humans could model in vivo liver-directed AAV expression of valoctocogene roxaparvovec (AAV5-hFVIII-SQ), an experimental gene therapy for hemophilia A with a hepatocyte-selective promoter. Relative levels of DNA and RNA in hepatocytes grown in vitro correlated with in vivo liver transduction across species. Expression in NHP hepatocytes more closely reflected expression in human hepatocytes than in mouse hepatocytes. We used this hepatocyte culture model to assess transduction efficacy of a novel liver-directed AAV capsid across species and identified which of 3 different canine factor VIII vectors produced the most transgene expression. Results were confirmed in vivo. Further, we determined mechanisms mediating inhibition of AAV5-hFVIII-SQ expression by concomitant isotretinoin using primary human hepatocytes. These studies support using in vitro primary hepatocyte models to predict species translatability of liver-directed AAV gene therapy and improve mechanistic understanding of drug-drug interactions.

AAV capsid design: A Goldilocks challenge.

In vivo therapeutic gene transfer has emerged as a novel class of medicines. Its feasibility relies on the safe and efficacious delivery of genetic cargo to the appropriate targets. The adeno-associated virus (AAV) vector manifested itself as a preferred gene delivery vehicle enabling therapeutic gene expression for several clinical indications. Here, we cover the recent trends in AAV capsid engineering to enhance its targeting specificity, safety, and endurance. While each and every desirable trait can be individually remodeled, combining several attributes in one capsid amounts to a significant engineering challenge. Taking advantage of virion structure and phylogenetics, harnessing directed evolution, sequence analyses, and machine learning, researchers develop novel capsid variants to realize the goals of safe and enduring gene therapy.

Interrogation of the Structure-Activity Relationship of a Lipophilic Nitroaromatic Prodrug Series Designed for Cancer Gene Therapy Applications.

PR-104A is a dual hypoxia/nitroreductase gene therapy prodrug by virtue of its ability to undergo either one- or two-electron reduction to its cytotoxic species. It has been evaluated extensively in pre-clinical GDEPT studies, yet off-target human aldo-keto reductase AKR1C3-mediated activation has limited its use. Re-evaluation of this chemical scaffold has previously identified SN29176 as an improved hypoxia-activated prodrug analogue of PR-104A that is free from AKR1C3 activation. However, optimization of the bystander effect of SN29176 is required for use in a GDEPT setting to compensate for the non-uniform distribution of therapeutic gene transfer that is often observed with current gene therapy vectors. A lipophilic series of eight analogues were synthesized from commercially available 3,4-difluorobenzaldehyde. Calculated octanol-water partition coefficients (LogD7.4) spanned > 2 orders of magnitude. 2D anti-proliferative and 3D multicellular layer assays were performed using isogenic HCT116 cells expressing E. coli NfsA nitroreductase (NfsA_Ec) or AKR1C3 to determine enzyme activity and measure bystander effect. A variation in potency for NfsA_Ec was observed, while all prodrugs appeared AKR1C3-resistant by 2D assay. However, 3D assays indicated that increasing prodrug lipophilicity correlated with increased AKR1C3 activation and NfsA_Ec activity, suggesting that metabolite loss from the cell of origin into media during 2D monolayer assays could mask cytotoxicity. Three prodrugs were identified as bono fide AKR1C3-negative candidates whilst maintaining activity with NfsA_Ec. These were converted to their phosphate ester pre-prodrugs before being taken forward into in vivo therapeutic efficacy studies. Ultimately, 2-(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-2-nitrobenzamido)ethyl dihydrogen phosphate possessed a significant 156% improvement in median survival in mixed NfsA_Ec/WT tumors compared to untreated controls (p = 0.005), whilst still maintaining hypoxia selectivity comparable to PR-104A.

Approaches towards biomaterial-mediated gene editing for cancer immunotherapy.

Gene therapies are transforming treatment modalities for many human diseases and disorders, including those in ophthalmology, oncology, and nephrology. To maximize the clinical efficacy and safety of these treatments, consideration of both delivery materials and cargos is critical. In consideration of the former, a large effort has been placed on transitioning away from potentially immunoreactive and toxic viral delivery mechanisms towards safer and highly tunable nonviral delivery mechanisms, including polymeric, lipid-based, and inorganic carriers. This change of paradigm does not come without obstacles, as efficient non-viral delivery is challenging, particularly to immune cells, and has yet to see clinical translation breakthroughs for gene editing. This mini-review describes notable examples of biomaterial-based gene delivery to immune cells, with emphasis on recent in vivo successes. In consideration of delivery cargos, clustered regularly interspaced palindromic repeat (CRISPR) technology is reviewed and its great promise in the field of immune cell gene editing is described. This mini-review describes how leading non-viral delivery materials and CRISPR technology can be integrated together to advance its clinical potential for therapeutic gene transfer to immune cells to treat cancer.

Discovery and validation of human genomic safe harbor sites for gene and cell therapies

SummaryExisting approaches to therapeutic gene transfer are marred by the transient nature of gene expression following non-integrative gene delivery and by safety concerns due to the random mechanism of viral-mediated genomic insertions. The disadvantages of these methods encourage future research in identifying human genomic sites that allow for durable and safe expression of genes of interest. We conducted a bioinformatic search followed by the experimental characterization of human genomic sites, identifying two that demonstrated the stable expression of integrated reporter and therapeutic genes without malignant changes to the cellular transcriptome. The cell-type agnostic criteria used in our bioinformatic search suggest widescale applicability of identified sites for engineering of a diverse range of tissues for clinical and research purposes, including modified T cells for cancer therapy and engineered skin to ameliorate inherited diseases and aging. In addition, the stable and robust levels of gene expression from identified sites allow for the industry-scale biomanufacturing of proteins in human cells.

Improved targeting of human CD4+ T cells by nanobody-modified AAV2 gene therapy vectors.

Adeno-associated viruses (AAV) are considered non-pathogenic in humans, and thus have been developed into powerful vector platforms for in vivo gene therapy. Although the various AAV serotypes display broad tropism, frequently infecting multiple tissues and cell types, vectors for specific and efficient targeting of human CD4+ T lymphocytes are largely missing. In fact, a substantial translational bottleneck exists in the field of therapeutic gene transfer that would require in vivo delivery into peripheral disease-related lymphocytes for subsequent genome editing. To solve this issue, capsid modification for retargeting AAV tropism, and in turn improving vector potency, is considered a promising strategy. Here, we genetically modified the minor AAV2 capsid proteins, VP1 and VP2, with a set of novel nanobodies with high-affinity for the human CD4 receptor. These novel vector variants demonstrated improved targeting of human CD4+ cells, including primary human peripheral blood mononuclear cells (PBMC) and purified human CD4+ T lymphocytes. Thus, the technical approach presented here provides a promising strategy for developing specific gene therapy vectors, particularly targeting disease-related peripheral blood CD4+ leukocytes.

In Situ Deployment of Engineered Extracellular Vesicles into the Tumor Niche via Myeloid-Derived Suppressor Cells.

Extracellular vesicles (EVs) have emerged as a promising carrier system for the delivery of therapeutic payloads in multiple disease models, including cancer. However, effective targeting of EVs to cancerous tissue remains a challenge. Here, it is shown that nonviral transfection of myeloid-derived suppressor cells (MDSCs) can be leveraged to drive targeted release of engineered EVs that can modulate transfer and overexpression of therapeutic anticancer genes in tumor cells and tissue. MDSCs are immature immune cells that exhibit enhanced tropism toward tumor tissue and play a role in modulating tumor progression. Current MDSC research has been mostly focused on mitigating immunosuppression in the tumor niche; however, the tumor homing abilities of these cells present untapped potential to deliver EV therapeutics directly to cancerous tissue. In vivo and ex vivo studies with murine models of breast cancer show that nonviral transfection of MDSCs does not hinder their ability to home to cancerous tissue. Moreover, transfected MDSCs can release engineered EVs and mediate antitumoral responses via paracrine signaling, including decreased invasion/metastatic activity and increased apoptosis/necrosis. Altogether, these findings indicate that MDSCs can be a powerful tool for the deployment of EV-based therapeutics to tumor tissue.

A novel Pah-exon1 deleted murine model of phenylalanine hydroxylase (PAH) deficiency

Phenylalanine hydroxylase (PAH) deficiency, colloquially known as phenylketonuria (PKU), is among the most common inborn errors of metabolism and in the past decade has become a target for the development of novel therapeutics such as gene therapy. PAH deficient mouse models have been key to new treatment development, but all prior existing models natively express liver PAH polypeptide as inactive or partially active PAH monomers, which complicates the experimental assessment of protein expression following therapeutic gene, mRNA, protein, or cell transfer. The mutant PAH monomers are able to form hetero-tetramers with and inhibit the overall holoenzyme activity of wild type PAH monomers produced from a therapeutic vector. Preclinical therapeutic studies would benefit from a PKU model that completely lacks both PAH activity and protein expression in liver. In this study, we employed CRISPR/Cas9-mediated gene editing in fertilized mouse embryos to generate a novel mouse model that lacks exon 1 of the Pah gene. Mice that are homozygous for the Pah exon 1 deletion are viable, severely hyperphenylalaninemic, accurately replicate phenotypic features of untreated human classical PKU and lack any detectable liver PAH activity or protein. This model of classical PKU is ideal for further development of gene and cell biologics to treat PKU.

Genetics in Cartilage Lesions: Basic Science and Therapy Approaches.

Cartilage lesions have a multifactorial nature, and genetic factors are their strongest determinants. As biochemical and genetic studies have dramatically progressed over the past decade, the molecular basis of cartilage pathologies has become clearer. Several homeostasis abnormalities within cartilaginous tissue have been found, including various structural changes, differential gene expression patterns, as well as altered epigenetic regulation. However, the efficient treatment of cartilage pathologies represents a substantial challenge. Understanding the complex genetic background pertaining to cartilage pathologies is useful primarily in the context of seeking new pathways leading to disease progression as well as in developing new targeted therapies. A technology utilizing gene transfer to deliver therapeutic genes to the site of injury is quickly becoming an emerging approach in cartilage renewal. The goal of this work is to provide an overview of the genetic basis of chondral lesions and the different approaches of the most recent systems exploiting therapeutic gene transfer in cartilage repair. The integration of tissue engineering with viral gene vectors is a novel and active area of research. However, despite promising preclinical data, this therapeutic concept needs to be supported by the growing body of clinical trials.

Overexpression of bone morphogenetic protein 3 (BMP-3) via raav-mediated gene transfer on the osteochondrogenic fate of human bone marrow-derived mesenchymal stem cells

Purpose: Injured adult articular cartilage is not capable of reliably regenerating an original hyaline structure. To improve the intrinsic healing activities of damaged cartilage, administration of genetically modified bone marrow-derived mesenchymal stem cells (MSCs) in cartilage lesions is a potent approach to improve cartilage repair. Here, we explored the potential benefits of overexpressing the bone morphogenetic protein 3 (BMP-3) [1] via clinically relevant recombinant adeno-associated virus (rAAV) vectors upon the commitment of human MSCs (hMSCs) as future therapeutic platforms for cartilage repair. Methods: rAAV vectors were packaged, purified, and titrated as previously described [2]. rAAV-lacZ carries the E. coli β-galactosidase (lacZ) reporter gene and rAAV-hBMP-3 a human BMP-3 sequence (OriGene), both controlled by the CMV-IE promoter/enhancer [2]. Bone marrow aspirates were obtained from the distal femurs of donors undergoing total knee arthroplasty, washed, and the pellets obtained were resuspended in DMEM, 10% FCS, 1% penicillin-streptomycin, and FGF-2 (10 ng/ml) for selection by adherence of progenitor cells and characterization of stem cell surface markers [2]. Cells (passage 2) were transduced with rAAV (rAAV-hBMP-3 or rAAV-lacZ: 40 μl each vector) or let untreated and kept either in monolayer cultures in DMEM, 10% FBS or as high-density aggregate cultures (2 x 105 cells) in chondrogenic medium (DMEM high glucose, ITS+ Premix, 1 mM pyruvate, 37.5 μg/ml ascorbate 2-phosphate, 10-7 M dexamethasone, 10 ng/ml TGF-β) for up to 21 days. Expression of BMP-3 was monitored by immunohistochemistry or by ELISA (R&D Systems) [2]. Histological analyses were performed on fixed monolayer cultures (ALP staining) or on paraffin-embedded sections of the aggregate cultures (5 μμ) (toluidine blue staining) [2]. Total RNA was extracted using the RNeasy Protect Mini kit (Qiagen) and reverse transcription was carried out with the 1st Strand cDNA Synthesis kit (Qiagen). cDNA amplification was performed via SYBR Green real-time RT-PCR [2]. Ct values were obtained for each target gene (SOX9, COL10A1) and GAPDH as a control for normalization, and fold inductions (relative to control samples) were measured using the 2-ΔΔCt method [2]. Each condition was performed in duplicate in three independent experiments. A t-test was employed with p < 0.05 considered statistically significant. Results: Successful BMP-3 overexpression was observed both in hMSC monolayer and aggregate cultures by application of rAAV-hBMP-3 relative to the control conditions (lack of vector delivery, reporter rAAV-lacZ gene transfer) over the whole period of evaluation (Fig. 1). Application of rAAV-hBMP-3 significantly increased the levels of cell proliferation in hMSC monolayer and aggregate cultures relative to the control conditions (up to 1.3- and 1.4 difference, respectively, p ≤ 0.001) (Table 1). Chondrogenic differentiation was evidenced by intense toluidine blue staining in hMSC aggregate cultures after 21 days, especially when the cells were treated with rAAV-hBMP-3 (Fig. 2). Similar results were noted regarding the osteogenic differentiation of the cells over time (Fig. 2). A real-time RT-PCR analysis revealed a reduced hypertrophic differentiation of the cells following rAAV-hBMP-3 transduction relative to the control treatments (up to 5-fold difference in COL10A1 expression, p ≤ 0.001), probably due to increased chondrogenic SOX9 gene expression levels (up to 8-fold difference, p ≤ 0.001) (Fig. 3). Conclusions: Gene transfer of BMP-3 via rAAV directs the osteochondrogenic commitment of hMSCs with a delay in hypertrophic differentiation. These Results demonstrate the possibility of modifying hMSCs by therapeutic BMP-3 gene transfer as a future, potential tool to enhance the repair processes in sites of osteochondral lesions. REFERENCES: [1] Wozney et al., Science 1988, 242:1528; [2] Venkatesan et al., Stem Cell Res Ther 2012, 3:22. ACKNOWLEDGMENTS: Work funded by the Deutsche Arthrose-Hilfe. The authors wish to thank Mary B. Goldring for helpful discussion.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Adenovirus vectors in hematopoietic stem cell genome editing.

Genome editing of hematopoietic stem cells (HSCs) represents a therapeutic option for a number of hematological genetic diseases, as HSCs have the potential for self-renewal and differentiation into all blood cell lineages. This review presents advances of genome editing in HSCs utilizing adenovirus vectors as delivery vehicles. We focus on capsid-modified, helper-dependent adenovirus vectors that are devoid of all viral genes and therefore exhibit an improved safety profile. We discuss HSC genome engineering for several inherited disorders and infectious diseases including hemoglobinopathies, Fanconi anemia, hemophilia, and HIV-1 infection by exvivo and invivo editing in transgenic mice, nonhuman primates, as well as in human CD34+ cells. Mechanisms of therapeutic gene transfer including episomal expression of designer nucleases and base editors, transposase-mediated random integration, and targeted homology-directed repair triggered integration into selected genomic safe harbor loci are also reviewed.

Pathophysiology of Calcium Mediated Ventricular Arrhythmias and Novel Therapeutic Options with Focus on Gene Therapy.

Cardiac arrhythmias constitute a major health problem with a huge impact on mortality rates and health care costs. Despite ongoing research efforts, the understanding of the molecular mechanisms and processes responsible for arrhythmogenesis remains incomplete. Given the crucial role of Ca2+-handling in action potential generation and cardiac contraction, Ca2+ channels and Ca2+ handling proteins represent promising targets for suppression of ventricular arrhythmias. Accordingly, we report the different roles of Ca2+-handling in the development of congenital as well as acquired ventricular arrhythmia syndromes. We highlight the therapeutic potential of gene therapy as a novel and innovative approach for future arrhythmia therapy. Furthermore, we discuss various promising cellular and mitochondrial targets for therapeutic gene transfer currently under investigation.

Molecular Engineering of Adeno-Associated Virus Capsid Improves Its Therapeutic Gene Transfer in Murine Models of Hemophilia and Retinal Degeneration.

Recombinant adeno-associated virus (AAV)-based gene therapy has been promising, but several host-related transduction or immune challenges remain. For this mode of therapy to be widely applicable, it is crucial to develop high transduction and permeating vectors that infect the target at significantly low doses. Because glycosylation of capsid proteins is known to be rate limiting in the life cycle of many viruses, we reasoned that perturbation of glycosylation sites in AAV2 capsid will enhance gene delivery. In our first set experiments, pharmacological modulation of the glycosylation status in host cells, modestly decreased (1-fold) AAV2 packaging efficacy while it improved their gene expression (~74%) in vitro. We then generated 24 mutant AAV2 vectors modified to potentially create or disrupt a glycosylation site in its capsid. Three of them demonstrated a 1.3–2.5-fold increase in transgene expression in multiple cell lines (HeLa, Huh7, and ARPE-19). Hepatic gene transfer of these vectors in hemophilia B mice, resulted in a 2-fold increase in human coagulation factor (F)IX levels, while its T/B-cell immunogenic response was unaltered. Subsequently, intravitreal gene transfer of glycosylation site-modified vectors in C57BL6/J mice demonstrated an increase in green fluorescence protein expression (~2- to 4-fold) and enhanced permeation across retina. Subretinal administration of these modified vectors containing RPE65 gene further rescued the photoreceptor response in a murine model of Leber congenital amarousis. Our studies highlight the translational potential of glycosylation site-modified AAV2 vectors for hepatic and ocular gene therapy applications.

Abstract 4772: Evaluation of immune response following ultrasound targeted gene therapy in a murine model of prostate cancer

Abstract Human Adenoviruses (hAd) have been broadly used as gene delivery tool in preclinical and clinical studies of cancer gene therapy. The main challenges associated with systemic delivery of hAds are their high immunogenicity and host-specificity. Adenoviruses can elicit strong innate and acquired immune responses that reduce therapeutic gene transfer and expression in malignant sites. Murine cells generally lack some of the receptors necessary for hAd adhesion and internalization making them not permissive to infection and replication and limiting translational studies using murine models. We have developed a gene transfer method, which uses lipid-encapsulated perfluorocarbon microbubbles (MBs) and ultrasound (US) to protect the hAds from the immune system and to deliver them to a targeted tissue, bypassing the necessity for specific receptors. The expression of the Ad-receptors (CAR, Integrins αvβ3 and αvβ5), transduction efficiency of Ad-GFP, and GFP transgene expression were measured in vitro in murine (TRAMP-C2) and human (DU145) prostate cancer cells lines by flow cytometry and immunofluorescence. The activation of the innate and acquired immune system following the injection of Ad-GFP/MBs complexes was evaluated in vivo in C57BL/6 mice healthy or bearing a TRAMP-C2 syngeneic tumor graft. ELISA was used to quantify the inflammatory cytokines TNF-α, IL-6 and measure Neutralizing Antibodies. ELISpot assay was performed to measure the frequency of DBP-specific CD8+ T cells secreting INF-γ. We showed in vitro that murine TRAMP-C2 cells display an expression pattern of Ad-receptors comparable to human DU145 prostate cancer cells. We also demonstrated that both murine and human cells showed a dose dependent increase in the percentage of cells transduced by Ad-GFP at 24 hours. Additionally, we showed that TRAMP-C2 cells efficiently express the GFP transgene at 48 and 72 hours post transduction. To assess in vivo if our gene transfer method could effectively protect the Ads form both the innate and adaptive immunity, we injected C57BL/6 mice with the hAds-GFP/MBs complexes +/-US. Notably we did not observe activation of either innate (secretion of TNF- and IL-6 cytokines) or acquired immunity (neutralizing antibodies and presence of adenovirus-specific CD8+ T cells producing INF-γ). Our data provides evidence that the TRAMP-C2 syngeneic model of prostate cancer is a suitable system to study in immunocompetent mice the capability of the MBs/US to protect the Adenoviruses from the immune system while delivering them to a targeted site bypassing the requirement of specific receptors. Citation Format: Flavia De Carlo, Elliot T. Varney, Pier Paolo Claudio, Candace M. Howard. Evaluation of immune response following ultrasound targeted gene therapy in a murine model of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4772.