Localized Transgene Expression Research Articles

Abstract CT213: A multicenter phase 1a/b study of NG-350A, a tumor-selective anti-CD40-antibody expressing adenoviral vector, and pembrolizumab in patients with metastatic or advanced epithelial tumors (FORTIFY)

Abstract Background: The efficacy of immune checkpoint inhibitors is often limited by immunosuppressive tumor microenvironments (TME) and novel combination therapies are required to overcome resistance. NG-350A is a novel T-SIGn (Tumor-Specific Immuno Gene) adenoviral vector that expresses a fully human agonistic IgG anti-CD40 antibody to promote innate and adaptive immune responses. Additionally, NG-350A selectively replicates in tumor cells, allowing IV dosing to be coupled with local transgene expression in the TME, thereby targeting all tumor lesions while limiting systemic exposure. Through these immunostimulatory effects the vector is designed to re-program ‘cold’ TMEs to allow functional anti-cancer immune responses. Data from an ongoing study with IV NG-350A monotherapy have shown promising tolerability, as well as prominent and sustained elevations in inflammatory cytokines (IL-2, IFNγ, IL-17A, IL-2 and IFNα2) consistent with the mechanism of action of anti-CD40 in stimulating TME re-programming [Naing 2021]. Based on these promising initial data, we designed a study to further assess the safety, tolerability and preliminary efficacy of NG-350A + pembrolizumab. Methods: FORTIFY (NCT05165433) is an open-label, dose-escalating, phase 1a/b study of NG-350A + pembrolizumab. Eligible patients have advanced/metastatic epithelial tumors that have progressed after ≥1 line of systemic therapy and are incurable by local therapy. Patients eligible for phase 1a must have experienced failure of prior PD-1/PD-L1 inhibition as part of any prior line of therapy; patients eligible for phase 1b must have primary resistance to PD-1/PD-L1 inhibition. During phase 1a, up to 30 patients will receive escalating doses of IV NG-350A (Bayesian Optimal Interval design) to a maximum of 1 × 1012 viral particles (vp) on Day 1 and 1 × 1013 vp on Days 3 and 5 (1 cycle). Patients will receive a fixed-dose of pembrolizumab (200 mg IV) on Day 15 and then every 3 weeks thereafter for up to 35 cycles. Phase 1b will further investigate the efficacy/safety of the selected regimen in up to 3 tumor-specific cohorts using a Simon 2-stage design. Co-primary objectives are to characterize the safety and tolerability of NG-350A + pembrolizumab and to identify a recommended dose. Preliminary efficacy and immunogenicity are secondary endpoints. Pharmacodynamic outcomes will be assessed using tumor tissues and blood. Analyses of tumor tissue (serial biopsies at baseline and Day 15 of cycles 1-3 [cycles 1-2 only in Phase 1b]) will explore virus replication, transgene expression and immune/inflammatory responses. Analyses of serial blood samples will explore cytokine production and changes in peripheral immune cell subsets. Recruitment is expected to begin in Q1 2022. Citation Format: Tom Lillie, Mark O'Hara, Christian Ottensmeier, Eileen Parkes, Lee Rosen, David Krige, Marya Chaney, Jo Carter, Vladimir Evilevitch, Matthew Thomas, Aung Naing. A multicenter phase 1a/b study of NG-350A, a tumor-selective anti-CD40-antibody expressing adenoviral vector, and pembrolizumab in patients with metastatic or advanced epithelial tumors (FORTIFY) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT213.

Abstract CT214: A multicenter phase 1a/b study of NG-641, a tumor-selective transgene-expressing adenoviral vector, and nivolumab in patients with metastatic or advanced epithelial tumors (NEBULA)

Abstract Background: T-SIGn (Tumor-Specific Immuno Gene Therapy) vectors are transgene-expressing variants of the blood-stable adenovirus enadenotucirev. Through a novel multimodal combination of immunostimulatory effects the vectors are designed to re-program ‘cold’ tumor microenvironments (TME) to allow functional anti-cancer immune responses. T-SIGn vectors selectively replicate in tumor cells, allowing IV delivery to be coupled with local transgene expression in the TME, thereby targeting all tumor lesions while limiting systemic exposure. NG-641 is a T-SIGn vector that expresses a fibroblast activation protein-directed bi-specific T-cell activator antibody to target cancer-associated fibroblasts (CAFs) and an immune-enhancer module (CXCL9/CXCL10/IFNα2) to recruit and activate immune cells. In an ongoing study (NCT04053283), NG-641 monotherapy has been successfully dose-escalated to 1 × 1012 viral particles (vp) on Day 1 and 3 × 1012 vp on Days 3 and 5; initial results from this study have shown a manageable tolerability profile and elevations in serum cytokines indicative of T-cell activation. Based on these promising initial data with NG-641 monotherapy, we designed a new study to assess NG-641 + nivolumab. Methods: NEBULA (NCT05043714) is an open-label, dose-escalating, phase 1a/b study of NG-641 + nivolumab. Eligible patients (pts) have advanced/metastatic epithelial tumors that have progressed after ≥1 line of systemic therapy and are incurable by local therapy. Pts eligible for phase 1a must have received prior PD-1/PD-L1 inhibition as part of any line of therapy; pts eligible for phase 1b must have primary resistance to PD-1/PD-L1 inhibition. During phase 1a, up to 30 pts will receive escalating doses of IV NG-641 (Bayesian Optimal Interval design) to a maximum dose of 1 × 1012 viral particles (vp) on Day 1 and 1 × 1013 vp on Days 3 and 5 (1 cycle). Pts will receive a fixed-dose of nivolumab (480 mg IV) on Day 15 and then every 4 weeks thereafter for up to 8 cycles. Phase 1b will further investigate the selected dose regimen in up to 3 tumor-specific cohorts (Cohorts A, B and C) using a Simon 2-stage design. Co-primary objectives are to characterize the safety and tolerability of NG-641 + nivolumab and to identify a recommended dose. Preliminary efficacy and immunogenicity are secondary endpoints. Pharmacodynamic outcomes will be assessed using tumor tissues and blood. Analyses of tumor tissue (serial biopsies at baseline and Day 15 of cycles 1-3 [cycles 1-2 only in Phase 1b]) will explore virus replication, transgene expression, immune/inflammatory responses and effects on CAFs by immunohistochemistry and gene expression analysis. Analyses of serial blood samples will explore cytokine production and changes in peripheral immune cell subsets. Enrollment to the first dose-escalation cohort is ongoing. Citation Format: Tom Lillie, Eileen Parkes, Christian Ottensmeier, David Krige, Behnaz Ravanfar, Vladimir Evilevitch, Matthew Thomas, Lee Rosen. A multicenter phase 1a/b study of NG-641, a tumor-selective transgene-expressing adenoviral vector, and nivolumab in patients with metastatic or advanced epithelial tumors (NEBULA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT214.

NEBULA: A multicenter phase 1a/b study of a tumor-selective transgene-expressing adenoviral vector, NG-641, and nivolumab in patients with metastatic or advanced epithelial tumors.

TPS2682 Background: Tumor-Specific Immuno Gene Therapy (T-SIGn) vectors are next-generation transgene-armed variants of the adenoviral vector enadenotucirev that selectively replicate in epithelial tumor cells. T-SIGn vectors are blood-stable, allowing IV delivery to be coupled with local transgene expression in the tumor microenvironment (TME), thereby targeting all lesions while limiting systemic exposure. The T-SIGn vector NG-641 encodes four immunostimulatory transgenes: fibroblast activation protein-directed bi-specific T-cell activator antibody to target cancer-associated fibroblasts (CAFs), IFNα2 to promote innate and adaptive immune responses, and CXCL9/10 to induce T-cell infiltration. Through this novel multimodal combination of immunostimulatory effects NG-641 is designed to re-program the TME to allow functional anti-cancer immune responses. In the ongoing STAR study (NCT04053283), NG-641 has been successfully dose-escalated to 1 × 1012 viral particles (vp) on Day 1 and 3 × 1012 vp on Days 3 and 5, with promising preliminary safety/tolerability and pharmacodynamic results. Based on these encouraging preliminary data with NG-641 monotherapy, we designed a new study to assess NG-641 + nivolumab. Methods: NEBULA (NCT05043714) is an open-label, dose-escalating, phase 1a/b study of NG-641 + nivolumab in patients (pts) with advanced/metastatic epithelial tumors that have progressed after ≥1 line of systemic therapy and are incurable by local therapy. Pts are eligible for phase 1a if they have received prior PD-1/PD-L1 inhibition as part of any line of therapy. During phase 1a, up to 30 pts will receive escalating doses of IV NG-641 to a maximum dose of 1 × 1012 viral particles (vp) on Day 1 and 1 × 1013 vp on Days 3 and 5 (1 cycle; Bayesian Optimal Interval design). Pts will receive a fixed-dose of nivolumab (480 mg IV) on Day 15 and then every 4 weeks thereafter for up to 8 cycles. In phase 1b, the recommended dose regimen will be further studied in patients with primary resistance to PD-1/PD-L1 inhibition; patients will be enrolled in up to 3 tumor-specific cohorts (Cohorts A-C; Simon 2-stage design). Co-primary objectives are to characterize the safety and tolerability of NG-641 + nivolumab and to identify a recommended dose. Preliminary efficacy and immunogenicity are secondary endpoints. Pharmacodynamic outcomes will also be assessed. Viral replication, transgene expression, immune/inflammatory responses and effects on CAFs by IHC and gene expression analysis will be analyzed using tumor tissue from serial biopsies (taken at baseline and Day 15 of cycles 1-3 [cycles 1-2 only in Phase 1b]). Serial blood samples will be analyzed to study cytokine production and changes in peripheral immune cell subsets. Enrollment to the first dose-escalation cohort is ongoing. Clinical trial information: NCT05043714.

NK Cell Effector Functions and Bystander Tumor Cell Killing in Immunovirotherapy.

Oncolytic virotherapy is a compelling strategy to combine cancer gene therapy with immunotherapy. Lytic virus replication in malignant cells not only enables localized transgene expression based on engineered vectors but also triggers immunogenic tumor cell death and elicits inflammation in the tumor microenvironment. Modified oncolytic viruses encoding immunomodulators have been developed to enhance antitumor immune effects and therapeutic efficacy. As one example, bispecific molecules that engage immune cells to exert antitumor cytotoxicity can be encoded within the viral vector. This chapter describes an in vitro coculture experiment to study functionality and antitumor efficacy of engineered measles vaccine strain virus encoding natural killer cell engagers. In a flow cytometry-based analysis, target cell death of noninfected bystander cancer cells and effector functions of primary human natural killer cells are investigated. This methodology can facilitate assessment of advanced oncolytic viral vectors for cancer immunotherapy.

Local transgene expression and whole-body transgenesis to model brain diseases in nonhuman primate.

Animal model is an essential tool in the life sciences research, notably in understanding the pathogenesis of the diseases and for further therapeutic intervention success. Rodents have been the most frequently used animals to model human disease since the establishment of gene manipulation technique. However, they remain inadequate to fully mimic the pathophysiology of human brain disease, partially due to huge differences between rodents and humans in terms of anatomy, brain function, and social behaviors. Nonhuman primates are more suitable in translational perspective. Thus, genetically modified animals have been generated to investigate neurologic and psychiatric disorders. The classical transgenesis technique is not efficient in that model; so, viral vector‐mediated transgene delivery and the new genome‐editing technologies have been promoted. In this review, we summarize some of the technical progress in the generation of an ad hoc animal model of brain diseases by gene delivery and real transgenic nonhuman primate.

Scaffold-mediated delivery for non-viral mRNA vaccines.

mRNA is increasingly being recognized as a promising alternative to pDNA in gene vaccinations. Only recently, owing to the needs of cancer immunotherapies, has the biomaterials/gene delivery community begun to develop new biomaterial strategies for immunomodulation. Here, we report a novel way to use implantable porous scaffolds as a local gene delivery depot to enhance mRNA vaccine immunization in vitro, and in vivo when compared with conventional bolus injections. We first evaluated transfection efficiencies of single-stranded mRNA condensed and charge neutralized with two lipids (Lipofectamine Messenger MAXTM LM-MM and StemfectTM SF) and two cationic polymers (in vivo-jetPEI™, Poly (β-amino ester)) as gene carriers. As SF demonstrated highest in vitro transfection and cell viability, it was selected for subsequent porous polymer scaffold-loading trials. Enhanced in vitro transfection of SF:mRNA nanoparticle-loaded poly (2-hydroxyethyl methacrylate) (pHEMA) scaffolds was also observed with a DC2.4 cell line. Improved sustained local release and local transgene expression were also demonstrated with SF:mRNA nanoparticle-loaded pHEMA scaffolds in vivo compared with bolus injections. Our results suggest that mRNA polyplex-loaded scaffolds may be a superior alternative to either repeated bolus immunizations or ex vivo transfection cell immunotherapies.

Localized lentivirus delivery via peptide interactions.

Gene delivery from biomaterial scaffolds has been employed to induce the expression of tissue inductive factors for applications in regenerative medicine. The delivery of viral vectors has been described as reflecting a balance between vector retention and release. Herein, we investigated the design of hydrogels in order to retain the vector at the material in order to enhance transgene expression. Poly(ethylene-glycol) (PEG) hydrogels were modified with poly-l-lysine (PLL) to non-covalently bind lentivirus. For cells cultured on the hydrogels, increasing the PLL molecular weight from 1 to 70 kDa led to increased transgene expression. The incubation time of the virus with the hydrogel and the PLL concentration modulated the extent of virus adsorption, and adsorbed virus had a 20% increase in the half-life at 37°C. Alternatives to high molecular weight PLL were identified through phage display technology, with peptide sequences specific for the VSV-G ectodomain, an envelope protein pseudotyped on the virus. These affinity peptides could easily be incorporated into the hydrogel, and expression was increased 20-fold relative to control peptide, and comparable to levels observed with the high molecular weight PLL. The modification of hydrogels with affinity proteins or peptides to bind lentivirus can be a powerful strategy to enhance and localized transgene expression. Biotechnol. Bioeng. 2016;113: 2033-2040. © 2016 Wiley Periodicals, Inc.

Mesenchymal stem cell-based gene therapy for erectile dysfunction.

Despite the overwhelming success of PDE5 inhibitor (PDE5I), the demand for novel pharmacotherapeutic and surgical options for ED continues to rise owing to the increased proportion of elderly individuals in the population, in addition to the growing percentage of ED patients who do not respond to PDE5I. Surgical treatment of ED is associated with many complications, thus warranting the need for nonsurgical therapies. Moreover, none of the above-mentioned treatments essentially corrects, cures or prevents ED. Although gene therapy is a promising option, many challenges and obstacles such as local inflammatory response and random transgene expression, in addition to other safety issues, limit its use at the clinical level. The use of stem cell therapy alone also has many shortcomings. To overcome these inadequacies, many scientists and clinicians are investigating new gene and stem cell therapies.

Targeted gene transfer to the brain via the delivery of brain-penetrating DNA nanoparticles with focused ultrasound

Gene therapy holds promise for the treatment of many pathologies of the central nervous system (CNS), including brain tumors and neurodegenerative diseases. However, the delivery of systemically administered gene carriers to the CNS is hindered by both the blood–brain barrier (BBB) and the nanoporous and electrostatically charged brain extracelluar matrix (ECM), which acts as a steric and adhesive barrier. We have previously shown that these physiological barriers may be overcome by, respectively, opening the BBB with MR image-guided focused ultrasound (FUS) and microbubbles and using highly compact “brain penetrating” nanoparticles (BPN) coated with a dense polyethylene glycol corona that prevents adhesion to ECM components. Here, we tested whether this combined approach could be utilized to deliver systemically administered DNA-bearing BPN (DNA-BPN) across the BBB and mediate localized, robust, and sustained transgene expression in the rat brain. Systemically administered DNA-BPN delivered through the BBB with FUS led to dose-dependent transgene expression only in the FUS-treated region that was evident as early as 24h post administration and lasted for at least 28days. In the FUS-treated region ~42% of all cells, including neurons and astrocytes, were transfected, while less than 6% were transfected in the contralateral non-FUS treated hemisphere. Importantly, this was achieved without any sign of toxicity or astrocyte activation. We conclude that the image-guided delivery of DNA-BPN with FUS and microbubbles constitutes a safe and non-invasive strategy for targeted gene therapy to the brain.

A next step in adeno‐associated virus‐mediated gene therapy for neurological diseases: regulation and targeting

Recombinant adeno-associated virus (rAAV) vectors mediating long term transgene expression are excellent gene therapy tools for chronic neurological diseases. While rAAV2 was the first serotype tested in the clinics, more efficient vectors derived from the rh10 serotype are currently being evaluated and other serotypes are likely to be tested in the near future. In addition, aside from the currently used stereotaxy-guided intraparenchymal delivery, new techniques for global brain transduction (by intravenous or intra-cerebrospinal injections) are very promising. Various strategies for therapeutic gene delivery to the central nervous system have been explored in human clinical trials in the past decade. Canavan disease, a genetic disease caused by an enzymatic deficiency, was the first to be approved. Three gene transfer paradigms for Parkinson's disease have been explored: converting L-dopa into dopamine through AADC gene delivery in the putamen; synthesizing GABA through GAD gene delivery in the overactive subthalamic nucleus and providing neurotrophic support through neurturin gene delivery in the nigro-striatal pathway. These pioneer clinical trials demonstrated the safety and tolerability of rAAV delivery in the human brain at moderate doses. Therapeutic effects however, were modest, emphasizing the need for higher doses of the therapeutic transgene product which could be achieved using more efficient vectors or expression cassettes. This will require re-addressing pharmacological aspects, with attention to which cases require either localized and cell-type specific expression or efficient brain-wide transgene expression, and when it is necessary to modulate or terminate the administration of transgene product. The ongoing development of targeted and regulated rAAV vectors is described.

3D-fection: Cell Transfection Within 3D Scaffolds and Hydrogels

3D matrices are widely used as cell growth supports in basic research, regenerative medicine or cell-based drug assays. In order to genetically manipulate cells cultured within 3D matrices, two novel non-viral transfection reagents allowing preparation of matrices for in situ cell transfection were evaluated. Two lipidic formulations, 3D-Fect™ and 3D-FectIN™, were assessed for their ability to transfect cells cultured within 3D solid scaffolds and 3D hydrogels, respectively. These reagents showed good compatibility with the most widespread types of matrices and enabled transfection of a wide range of mammalian cells of various origins. Classical cell lines, primary cells and stem cells were thus genetically modified while colonizing their growth support. Importantly, this in situ strategy alleviated the need to manipulate cells before seeding them. Results presented here demonstrated that 3D-Fect and 3D-FectIN reagents for 3D transfection are totally compatible with cells and do not impair matrix properties. 3D-Fect and 3D-FectIN, therefore, provide valuable tools for achieving localized and sustained transgene expression and should find versatile applications in fundamental research, regenerative medicine and cell-based drug assays.

Gene Delivery Strategies Targeting Stable Atheromatous Plaque

Conventional therapeutic options to treat chronic angina pectoris are pharmacological interventions, coronary bypass surgery (CABG) and percutaneous coronary intervention (PCI). In animal models, it was shown that gene delivery strategies harbour an exciting potential to support and maybe even replace conventional anti-angina treatments, but the translation of the basic science to clinical practise appears problematic. Gene therapy targeting key elements of neointima formation (e.g. cell cycle regulators, metalloproteinases, inflammation and oxidative stress) reduces vein graft and stent failure in experimental models. Additionally, systemic gene delivery of genes targeting NO production, oxidative stress, inflammation and foam cell formation has been shown to prevent atherosclerosis in different animal models. During CABG the vein graft can be transfected ex vivo and during PCI, a stent carrying transfection vectors can be deployed. Both strategies result in the induction of local transgene expression at the site of interest. This limits unwarranted transgene expression and the toxicity seen with systemic gene delivery. However, with the development of new transfection vectors, able to induce local transgene expression without detrimental side effects, systemic anti-inflammatory and anti-oxidative, gene delivery could be a powerful tool in secondary prevention.

IL10 Released by a New Inflammation-regulated Lentiviral System Efficiently Attenuates Zymosan-induced Arthritis

Administration of anti-inflammatory cytokines is a common therapeutic strategy in chronic inflammatory diseases. Gene therapy is an efficient method for delivering therapeutic molecules to target cells. Expression of the cell adhesion molecule E-selectin (ESEL), which is expressed in the early stages of inflammation, is controlled by proinflammatory cytokines, making its promoter a good candidate for the design of inflammation-regulated gene therapy vectors. This study describes an ESEL promoter (ESELp)-based lentiviral vector (LV) that drives localized transgene expression during inflammation. Mouse matrigel plug assays with ESELp-transduced endothelial cells showed that systemic lipopolysaccharide (LPS) administration selectively induces ESELp-controlled luciferase expression in vivo. Inflammation-specific induction was confirmed in a mouse model of arthritis, showing that this LV is repeatedly induced early in acute inflammation episodes and is downregulated during remission. Moreover, the local acute inflammatory response in this animal model was efficiently blocked by expression of the anti-inflammatory cytokine interleukin-10 (IL10) driven by our LV system. This inflammation-regulated expression system has potential application in the design of new strategies for the local treatment of chronic inflammatory diseases such as cardiovascular and autoimmune diseases.

Sustained, localized transgene expression mediated from lentivirus‐loaded biodegradable polyester elastomers

The study of biomaterials for gene delivery in tissue engineering and regenerative medicine is a growing area, necessitating the investigation of new biomaterials and gene delivery vectors. Poly(1,8-octanediol citrate) (POC) and poly(glycerol-sebacate) (PGS) are biodegradable, biocompatible elastomers that have tunable mechanical properties, surface characteristics, and degradation rate. The objective of this work was to investigate whether POC and PGS would support the immobilization and release of lentivirus to allow sustained and localized transgene expression. Porous biomaterials were prepared using salt as a porogen, and in vitro and in vivo transgene expression from immobilized and released lentiviruses were assessed. Cells seeded onto biomaterials loaded with lentiviruses yielded titer-dependent transgene expression in vitro. Lentivirus activity on both biomaterials was maintained for at least 5 days. When implanted subcutaneously in rats, POC and PGS with immobilized lentivirus exhibited sustained and localized transgene expression for at least 5 weeks. This research demonstrates that lentivirus immobilization on POC and PGS is feasible and potentially useful for a variety of tissue engineering and regenerative medicine applications.

Fibrin hydrogels for lentiviral gene delivery in vitro and in vivo

Gene delivery from hydrogels represents a versatile approach for localized expression of tissue inductive factors that can promote cellular processes that lead to regeneration. Lentiviral gene therapy vectors were entrapped within fibrin hydrogels, either alone or complexed with hydroxylapatite (HA) nanoparticles. The inclusion of HA into the hydrogel led to the formation of small aggregates distributed throughout the hydrogel, with no obvious alteration of the pore structure outside the aggregates. The presence of HA slowed hydrogel degradation by collagenase and plasmin relative to fibrin alone, and also decreased the rate of cell migration. Lentivirus had similar release from the fibrin hydrogels formed with or without HA. The altered hydrogel properties suggest an interaction between the nanoparticle and fibrin, which may displace the virus from the particle leading to similar release profiles. Transgene expression by cells migrating into the hydrogel in vitro was reduced in the presence of HA, consistent with the role of cell migration on transgene expression. In vivo, lentivirus loaded fibrin hydrogels promoted localized transgene expression that increased through day 9 and decreased through day 14. For the fibrin only hydrogels, expression continued to decline after day 14. However, hydrogels with HA maintained this transgene expression level for an additional 2 weeks before declining. Immunostaining identified transgene primarily outside the fibrin-HA gel at day 9; however, at day 21, transgene expression was observed primarily within the fibrin-HA gel. The localized delivery of lentivirus provides an opportunity to enhance the bioactivity of fibrin hydrogels for a wide range of applications in regenerative medicine.

Gene-eluting stents: non-viral, liposome-based gene delivery of eNOS to the blood vessel wall in vivo results in enhanced endothelialization but does not reduce restenosis in a hypercholesterolemic model

Although successful, drug-eluting stents require significant periods of dual anti-platelet therapy with a persistent risk of late stent thrombosis due to inhibition of re-endothelialization. Endothelial regeneration is desirable to protect against in-stent thrombosis. Gene-eluting stents may be an alternative allowing inhibition of neointima and regenerating endothelium. We have shown that adenoviral endothelial nitric oxide synthase (eNOS) delivery can result in significantly decreased neointimal formation and enhanced re-endothelialization. Here, we examined non-viral reporter and therapeutic gene delivery from a stent. We coated lipoplexes directly onto the surface of stents. These lipostents were then deployed in the injured external iliac artery of either normal or hypercholesterolemic New Zealand White rabbits and recovered after 28 days. Lipoplexes composed of lipofectin and a reporter lacZ gene or therapeutic eNOS gene were used. We demonstrated efficient gene delivery at 28 days post-deployment in the media (21.3±7.5%) and neointima (26.8±11.2%). Liposomal delivery resulted in expression in macrophages between the stent struts. This resulted in improved re-endothelialization as detected by two independent measures compared with vector and stent controls (P<0.05 for both). However, in contrast to viral delivery of eNOS, liposomal eNOS does not reduce restenosis rates. The differing cell populations targeted by lipoplexes compared with adenoviral vectors may explain their ability to enhance re-endothelialization without affecting restenosis. Liposome-mediated gene delivery can result in prolonged and localized transgene expression in the blood vessel wall in vivo. Furthermore, lipoeNOS delivery to the blood vessel wall results in accelerated re-endothelialization; however, it does not reduce neointimal formation.

Lentivirus Immobilization to Nanoparticles for Enhanced and Localized Delivery From Hydrogels

Hydrogels can provide a controllable cell microenvironment for numerous applications in regenerative medicine, and delivery of gene therapy vectors can be employed to enhance their bioactivity. We investigated the delivery of lentiviral vectors from hydrogels, and employed the immobilization of lentivirus to hydroxylapatite (HA) nanoparticles as a means to retain and stabilize vectors within hydrogels, and thereby increase delivery efficiency. Entrapment of the vector alone within the hydrogel maintained the activity of the virus more effectively compared to the absence of a hydrogel, and release was slowed with an increasing solid content of the hydrogel. Association of the lentivirus with HA increased the activity of the complexes, with HA increasing the virus activity for 72 hours. Cells seeded onto lentivirus-HA-loaded hydrogels had a decreased number of infected cells outside of the hydrogel relative to the absence of HA. In vivo studies with collagen hydrogels loaded with lentivirus and HA-lentivirus demonstrated sustained and localized transgene expression for at least 4 weeks, with increased expression using the lentivirus-HA complex. This strategy of nanoparticle immobilization to stabilize and retain vectors is broadly applicable to hydrogels, and may provide a versatile tool to combine gene therapy and biomaterials for applications in regenerative medicine.

Route of Adenovirus-Based HIV-1 Vaccine Delivery Impacts the Phenotype and Trafficking of Vaccine-Elicited CD8 + T Lymphocytes

Candidate HIV-1 vaccine regimens utilizing intramuscularly (i.m.) administered recombinant adenovirus (rAd)-based vectors can induce potent mucosal cellular immunity. However, the degree to which mucosal rAd vaccine routing might alter the quality and anatomic distribution of vaccine-elicited CD8(+) T lymphocytes remains unclear. We show that the route of vaccination critically impacts not only the magnitude but also the phenotype and trafficking of antigen-specific CD8(+) T lymphocytes in mice. I.m. rAd immunization induced robust local transgene expression and elicited high-frequency, polyfunctional CD8(+) T lymphocytes that trafficked broadly to both systemic and mucosal compartments. In contrast, intranasal (i.n.) rAd immunization led to similarly robust local transgene expression but generated low-frequency, monofunctional CD8(+) T lymphocytes with restricted anatomic trafficking patterns. Respiratory rAd immunization elicited systemic and mucosal CD8(+) T lymphocytes with phenotypes and trafficking properties distinct from those elicited by i.m. or i.n. rAd immunization. Our findings indicate that the anatomic microenvironment of antigen expression critically impacts the phenotype and trafficking of antigen-specific CD8(+) T lymphocytes.

Adeno‐associated virus type 5–mediated intraarticular administration of tumor necrosis factor small interfering RNA improves collagen‐induced arthritis

RNA interference (RNAi) is a powerful tool for sequence-specific gene silencing, and interest in its application in human diseases is growing. Given the success of recent strategies for administering gene therapy in rheumatoid arthritis using recombinant vectors such as adeno-associated virus type 5 (rAAV5) for optimized intraarticular gene transfer, we undertook the present study to determine the feasibility of using rAAV5-mediated RNAi-based therapy in arthritis. We developed rAAV5 vectors expressing short hairpin small interfering RNA (shRNA) against tumor necrosis factor alpha (TNFalpha) under H1 promoter, and carrying the enhanced green fluorescent protein (eGFP) reporter gene under cytomegalovirus promoter (rAAV5-shTNF). TNFalpha gene silencing was validated in vitro with mouse macrophages. Mice with collagen-induced arthritis were injected in the ankle and knee joints, at disease onset, with either rAAV5-shTNF or control rAAV5-eGFP vectors (5 x 10(9) particles). Arthritis severity was assessed clinically and histologically, and immunologic response was examined. Local and systemic transgene expression was monitored using quantitative reverse transcriptase-polymerase chain reaction, immunohistochemical analysis, and enzyme-linked immunosorbent assay. After a single injection of rAAV5-shTNF into inflamed joints, local TNFalpha gene silencing provided rapid and long-term suppression of arthritis progression and reduced joint damage compared with that observed in control groups. Treatment with rAAV5-shTNF was associated with decreased proliferation and interferon-gamma production by antigen-stimulated T cells from draining lymph nodes, and the potency of this treatment was similar to that observed with other treatment strategies targeting TNFalpha at the protein level, either locally or systemically. Our data present the first proof-of-concept for the application of rAAV5-mediated RNAi-based gene therapy for local blockade of inflammation in experimental arthritis.

Local Gene Transfer and Expression Following Intramuscular Administration of FGF-1 Plasmid DNA in Patients With Critical Limb Ischemia

NV1FGF is an expression plasmid encoding sp.FGF-1(21-154) currently under investigation for therapeutic angiogenesis in clinical trials. NV1FGF plasmid distribution and transgene expression following intramuscular (IM) injection in patients is unknown. The study involved six patients with chronic critical limb ischemia (CLI) planned to undergo amputation. A total dose of 0.5, 2, or 4 mg NV1FGF was administered as eight IM injections (0.006, 0.25, or 0.5 mg per injection) 3-5 days before amputation. Injected sites (30 cm(3)) were divided into equally sized smaller pieces to assess spatial distribution of NV1FGF sequences (PCR), NV1FGF mRNA (reverse transcriptase-PCR), and fibroblast growth factor-1 (FGF-1)-expressing cells (immunohistochemistry). Data indicated gene expression at all doses. The distribution area was within 5-12 cm for NV1FGF sequences containing the expression cassette, up to 5 cm for NV1FGF mRNA, and up to 3 cm for FGF-1-expressing myofibers. All FGF receptors were detected indicating robust potential for bioactivity after NV1FGF gene transfer. Circulating levels of NV1FGF sequences were shown to decrease within days after injection. Data support demonstration of plasmid-mediated gene transfer and expression in muscles from patients with CLI. FGF-1 expression was shown to be limited to injection sites, which supports the concept of multiple-site injection for therapeutic use.