Therapeutic Cassette Research Articles

Stable transduction of the neonatal mouse liver using a hybrid rAAV/sleeping beauty transposon gene delivery system.

Conventional adeno-associated viral (AAV) vectors, while highly effective in quiescent cells such as hepatocytes in the adult liver, confer less durable transgene expression in proliferating cells owing to episome loss. Sustained therapeutic success is therefore less likely in liver disorders requiring early intervention. We have previously developed a hybrid, dual virion approach, recombinant AAV (rAAV)/piggyBac transposon system capable of achieving stable gene transfer in proliferating hepatocytes at levels many fold above conventional AAV vectors. An alternative transposon system, Sleeping Beauty, has been widely used for ex vivo gene delivery; however liver-targeted delivery using a hybrid rAAV/Sleeping Beauty approach remains relatively unexplored. We investigated the capacity of a Sleeping Beauty (SB)-based dual rAAV virion approach to achieve stable and efficient gene transfer to the newborn murine liver using transposable therapeutic cassettes encoding coagulation factor IX or ornithine transcarbamylase (OTC). At equivalent doses, rAAV/SB100X transduced hepatocytes with high efficiency, achieving stable expression into adulthood. Compared with conventional AAV, the proportion of hepatocytes transduced, and factor IX and OTC activity levels, were both markedly increased. The proportion of hepatocytes stably transduced increased 4- to 8-fold from <5%, and activity levels increased correspondingly, with markedly increased survival and stable urinary orotate levels in the OTC-deficient Spfash mouse following elimination of residual endogenous murine OTC. The present study demonstrates the first in vivo utility of a hybrid rAAV/SB100X transposon system to achieve stable long-term therapeutic gene expression following delivery to the highly proliferative newborn mouse liver. These results have relevance to the treatment of genetic metabolic liver diseases with neonatal onset.

A self-complementary AAV proviral plasmid that reduces cross-packaging and ITR promoter activity in AAV vector preparations

Adeno-associated viral vectors (AAV) are a leading delivery system for gene therapy in animal models and humans. With several FDA-approved AAV gene therapies on the market, issues related to vector manufacturing have become increasingly important. In this study, we focused on potentially toxic DNA contaminants that can arise from AAV proviral plasmids, the raw materials required for manufacturing recombinant AAV in eukaryotic cells. Typical AAV proviral plasmids are circular DNAs containing a therapeutic gene cassette flanked by natural AAV inverted terminal repeat (ITR) sequences, and a plasmid backbone carrying prokaryotic sequences required for plasmid replication and selection in bacteria. While the majority of AAV particles package the intended therapeutic payload, some capsids instead package the bacterial sequences located on the proviral plasmid backbone. Since ITR sequences also have promoter activity, potentially toxic bacterial open reading frames can be produced in vivo, thereby representing a safety risk. In this study, we describe a new AAV proviral plasmid for vector manufacturing that (1) significantly decreases cross-packaged bacterial sequences; (2) increases correctly packaged AAV payloads; and (3) blunts ITR-driven transcription of cross-packaged material to avoid expressing potentially toxic bacterial sequences. This system may help improve the safety of AAV vector products.

Hematopoietic stem cell gene editing rescues B-cell development in X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) is an inborn error of immunity that renders boys susceptible to life-threatening infections due to loss of mature B cells and circulating immunoglobulins. It is caused by defects in the gene encoding the Bruton tyrosine kinase (BTK) that mediates the maturation of B cells in the bone marrow and their activation in the periphery. This paper reports on a gene editing protocol to achieve "knock-in" of a therapeutic BTK cassette in hematopoietic stem and progenitor cells (HSPCs) as a treatment for XLA. To rescue BTK expression, this study employed a clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system that creates a DNA double-strand break in an early exon of the BTK locus and an adeno-associated virus 6 virus that carries the donor template for homology-directed repair. The investigators evaluated the efficacy of the gene editing approach in HSPCs from patients with XLA that were cultured invitro under B-cell differentiation conditions or that were transplanted in immunodeficient mice to study B-cell output invivo. A (feeder-free) B-cell differentiation protocol was successfully applied to blood-mobilized HSPCs to reproduce invitro the defects in B-cell maturation observed in patients with XLA. Using this system, the investigators could show the rescue of B-cell maturation by gene editing. Transplantation of edited XLA HSPCs into immunodeficient mice led to restoration of the human B-cell lineage compartment in the bone marrow and immunoglobulin production in the periphery. Gene editing efficiencies above 30% could be consistently achieved in human HSPCs. Given the potential selective advantage of corrected cells, as suggested by skewed X-linked inactivation in carrier females and by competitive repopulating experiments in mouse models, this work demonstrates the potential of this strategy as a future definitive therapy for XLA.

Bacteriophage-based particles carrying the TNF-related apoptosis-inducing ligand (TRAIL) gene for targeted delivery in hepatocellular carcinoma.

The TRAIL (Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand) is a promising candidate for cancer treatment due to its unique ability to selectively induce programmed cell death, or apoptosis, in cancer cells while sparing healthy ones. This selectivity arises from the preferential binding of the TRAIL to death receptors on cancer cells, triggering a cascade of events that lead to their demise. However, significant limitations in using the TRAIL for cancer treatment are the administration of the TRAIL protein that can potentially lead to tissue toxicity (off-target) and the short half-life of the TRAIL in the body which may necessitate frequent and sustained administration; these can pose logistical challenges for long-term treatment regimens. We have devised a novel approach for surmounting these limitations by introducing the TRAIL gene directly into cancer cells, enabling them to produce the TRAIL locally and subsequently trigger apoptosis. A novel gene delivery system such as a bacteriophage-based particle TPA (transmorphic phage/AAV) was utilized to address these limitations. TPA is a hybrid M13 filamentous bacteriophage particle encapsulating a therapeutic gene cassette with inverted terminal repeats (ITRs) from adeno-associated viruses (AAVs). The particle also showed a tumour targeting ligand, CDCRGDCFC (RGD4C), on its capsid (RGD4C.TPA) to target the particle to cancer cells. RGD4C selectively binds to αvβ3 and αvβ5 integrins overexpressed on the surface of most of the cancer cells but is barely present on normal cells. Hepatocellular carcinoma (HCC) was chosen as a model because it has one of the lowest survival rates among cancers. We demonstrated that human HCC cell lines (Huh-7 and HepG2) express αvβ5 integrin receptors on their surface. These HCC cells also express death receptors and TRAIL-binding receptors. We showed that the targeted TPA particle carrying the transmembrane TRAIL gene (RGD4C.TPA-tmTRAIL) selectively and efficiently delivered the tmTRAIL gene to HCC cells resulting in the production of tmTRAIL from transduced cells and subsequently induced apoptotic death of HCC cells. This tumour-targeted particle can be an excellent candidate for the targeted gene therapy of HCC.

Abstract 13927: AAV Gene Therapy to Treat Underlying Cardiac Biomechanical Defects in Lmna Dilated Cardiomyopathy Improves Lifespan and Preserves Ejection Fraction in Lmna DCM Mouse Model

Mutations in the LMNA gene are the second most common cause of genetic dilated cardiomyopathies (DCMs) with a prevalence of ~1/12500. Adeno-associated virus (AAV) gene therapies are a promising treatment modality for genetic disease. However, the autosomal dominant, gain-of-function mutations underlying LMNA DCM preclude standard gene replacement approaches. To understand LMNA DCM, we previously generated a mouse model that exhibits reduced ejection fraction, dilation of ventricular inner dimensions and increased cardiac fibrosis, surviving for only ~40 days after cardiac-specific deletion of the Lmna gene. We propose that the mutant LMNA gene weakens the structural integrity of the nucleus, making it vulnerable to biomechanical forces generated by contracting cardiomyocytes, which ultimately results in nuclear damage and a decline in cardiac function. Our gene therapy, AAV9-cTnT-GSLA01, prevents nuclear damage and disease in our Lmna DCM model by reducing biomechanical forces transmitted to the nucleus. To determine if we can treat, rather than prevent, Lmna DCM, we injected animals with AAV9-cTnT-GSLA01 at 1x10 14 vg/kg at different timepoints (21, 17, 14 and 1 day(s)) after induction of Lmna deletion. Treatment with AAV9-cTnT-GSLA01 17 days post Lmna deletion resulted in lifespan extension to 108 days (P= 0.0002), while earlier intervention at day 14 or day 1, improved lifespan to 122 and 208 days respectively (P = 0.0002, P= 0.0035). To address dose dependency of AAV9-cTnT-GSLA01, we delivered the vector at 5x10 13 vg/kg, 1x10 14 vg/kg, and 2x10 14 vg/kg at 17 days after Lmna deletion. Lifespan was extended from 40 days in untreated mice to 51.5 (P = 0.0135), 67.5 (P &lt; 0.0001), and 103.5 days (P &lt; 0.0001) respectively. The progressive decline of ejection fraction in these mice was attenuated in a dose-dependent manner. Thus, the positive effect of AAV9-cTnT-GSLA01 on lifespan and cardiac function in Lmna DCM mice is enhanced with increasing dose and transgene expression. Initial optimisation of the therapeutic expression cassette appears to improve lifespan extension significantly. AAV9-cTnT-GSLA01 is a novel gene therapy approach that promises to address the underlying cause of LMNA DCM.

Minicircle Biopharmaceuticals–An Overview of Purification Strategies

Minicircles are non-viral delivery vectors with promising features for biopharmaceutical applications. These vectors are plasmid-derived circular DNA molecules that are obtained in vivo in Escherichia coli by the intramolecular recombination of a parental plasmid, which generates a minicircle containing the eukaryotic therapeutic cassette of interest and a miniplasmid containing the prokaryotic backbone. The production process results thus in a complex mixture, which hinders the isolation of minicircle molecules from other DNA molecules. Several strategies have been proposed over the years to meet the challenge of purifying and obtaining high quality minicircles in compliance with the regulatory guidelines for therapeutic use. In minicircle purification, the characteristics of the strain and parental plasmid used have a high impact and strongly affect the purification strategy that can be applied. This review summarizes the different methods developed so far, focusing not only on the purification method itself but also on its dependence on the upstream production strategy used.

Targeted epigenetic repression by CRISPR/dSaCas9 suppresses pathogenic DUX4-fl expression in FSHD

Facioscapulohumeral muscular dystrophy (FSHD) is caused by incomplete silencing of the disease locus, leading to pathogenic misexpression of DUX4 in skeletal muscle. Previously, we showed that CRISPR inhibition could successfully target and repress DUX4 in FSHD myocytes. However, an effective therapy will require both efficient delivery of therapeutic components to skeletal muscles and long-term repression of the disease locus. Thus, we re-engineered our platform to allow in vivo delivery of more potent epigenetic repressors. We designed an FSHD-optimized regulatory cassette to drive skeletal muscle-specific expression of dCas9 from Staphylococcus aureus fused to HP1α, HP1γ, the MeCP2 transcriptional repression domain, or the SUV39H1 SET domain. Targeting each regulator to the DUX4 promoter/exon 1 increased chromatin repression at the locus, specifically suppressing DUX4 and its target genes in FSHD myocytes and in a mouse model of the disease. Importantly, minimizing the regulatory cassette and using the smaller Cas9 ortholog allowed our therapeutic cassettes to be effectively packaged into adeno-associated virus (AAV) vectors for in vivo delivery. By engineering a muscle-specific epigenetic CRISPR platform compatible with AAV vectors for gene therapy, we have laid the groundwork for clinical use of dCas9-based chromatin effectors in skeletal muscle disorders.

Abstract 3132: Therapeutic blockade of Foxp3 in experimental breast cancer: Immune stimulation and direct antitumor effects

Abstract Our previous results indicate that systemic administration of a cell penetrating peptide (P60) that inhibits Foxp3, a transcription factor required for Treg function, improves the efficacy of antitumor vaccines in experimental breast cancer. We also found that P60 exerts direct antitumor effects, inhibiting tumor progression in Foxp3+ breast tumors. Although there is controversy over the role of Foxp3 in tumor cells, we found that P60 inhibits survival, proliferation and release of IL-10 in Foxp3+ breast tumor cells. Here we aimed to evaluate the pathways that control Foxp3 expression in murine LM3 breast tumor cells and to develop gene therapy vectors encoding P60 in order to improve the availability of this peptide in vivo. We assessed the role of TGF-β, mTOR and prostaglandins on the expression of Foxp3 in breast tumor cells, as these pathways have been shown to modulate Foxp3 expression in Tregs. Stimulation of LM3 cells with TGF-β and mTOR inhibitor rapamicyn upregulated Foxp3 expression, as assessed by flow cytometry, whereas indomethacin, an inhibitor of prostaglandin synthase cyclooxygenase, inhibited Foxp3 expression. These observations suggest that the regulatory mechanisms of Foxp3 expression are similar in breast tumor cells and Tregs. We next developed a plasmid that encodes P60 linked through an IRES sequence to Td.Tomato as a reporter gene (pCMV.P60.dTomato), as well a control plasmid. Transduction efficiency of these plasmids was evaluated in murine 4T1 breast tumor cells, which exhibit low expression of Foxp3 and thus are not affected by P60. Expression of Td.Tomato was readily detected by fluorescence microscopy. Conditioned media from pCMV.P60.dTomato-transfected 4T1 cells reduced the proliferation and the secretion of IL-10 in Foxp3+ LM3 cells when compared to control plasmid-transfected cells, which suggests that functional P60 is released from pCMV.P60.dTomato-transfected cells. In order to develop a gene therapeutic strategy to deliver P60 in vivo, we developed an adenoviral vector (Ad.P60.dTomato) encoding the therapeutic cassette, as well as a control vector (Ad.dTomato). These vectors successfully transduced breast tumor cells in vitro and in vivo, as evaluated by TdTomato expression. Our findings indicate that P60 could be delivered using gene therapy vectors, which could be useful for the treatment of breast cancer. Citation Format: Alejandro J. Nicola, Mariela A. Moreno Ayala, Maria F. Gottardo, Antonela S. Asad, Camila F. Zuccato, ELISA Bal de Kier Joffé, Adriana Seilicovich, Flavia Zanetti, Marianela Candolfi. Therapeutic blockade of Foxp3 in experimental breast cancer: Immune stimulation and direct antitumor effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3132.

Homology-Directed Recombination for Enhanced Engineering of Chimeric Antigen Receptor T Cells

Gene editing by homology-directed recombination (HDR) can be used to couple delivery of a therapeutic gene cassette with targeted genomic modifications to generate engineered human T cells with clinically useful profiles. Here, we explore the functionality of therapeutic cassettes delivered by these means and test the flexibility of this approach to clinically relevant alleles. Because CCR5-negative T cells are resistant to HIV-1 infection, CCR5-negative anti-CD19 chimeric antigen receptor (CAR) T cells could be used to treat patients with HIV-associated B cell malignancies. We show that targeted delivery of an anti-CD19 CAR cassette to the CCR5 locus using a recombinant AAV homology template and an engineered megaTAL nuclease results in T cells that are functionally equivalent, in both in vitro and in vivo tumor models, to CAR T cells generated by random integration using lentiviral delivery. With the goal of developing off-the-shelf CAR T cell therapies, we next targeted CARs to the T cell receptor alpha constant (TRAC) locus by HDR, producing TCR-negative anti-CD19 CAR and anti-B cell maturation antigen (BCMA) CAR T cells. These novel cell products exhibited in vitro cytolytic activity against both tumor cell lines and primary cell targets. Our combined results indicate that high-efficiency HDR delivery of therapeutic genes may provide a flexible and robust method that can extend the clinical utility of cell therapeutics.

571. Safe Harbor Targeting of IL2RG Expression Cassettes for Gene Therapy of X-Linked Severe Combined Immunodeficiency (SCID-X1)

SCID-X1 is caused by mutations in the IL2RG gene and results in severe defects in T, B, and NK-cell mediated immunity. Transduction of hematopoietic stem and progenitor cells with IL2RG-expressing gamma-retroviral vectors can be clinically effective but has also caused leukemia in several trials due to vector integrations that activate T-cell proto-oncogenes. One approach to eliminate these genotoxic integrations is to use genome editing techniques to insert an IL2RG transgene into a genomic safe harbor loci. The choice of a genomic safe harbor rather than the endogenous IL2RG locus offers several advantages including correction of a wide variety of mutations with a single therapeutic cassette, potentially higher editing efficiency in selected safe harbor sites, and the ability to adapt the specific nuclease reagents to other diseases and therapeutic templates. We are testing two different IL2RG-expression cassettes for targeting and expression into the AAVS1 locus. First, we generated a recombination template that contains a codon-optimized IL2RG cDNA driven by the short elongation factor alpha (EF1a) promoter based on clinical data with this cassette being transmitted via a lentiviral vector and showing immune correction in human SCID-X1 subjects in an ongoing trial at the NIH Clinical Center. We first tested whether a sufficient level of IL2RG expression can be achieved when a single copy of the EF1α-IL2RG-cDNA cassette was inserted into the AAVS1 locus in human ED7R T-cells. This transgene cassette was flanked by 500bp homologous regions from the AAVS1 locus and transfected into ED7R cells, along with a pair of TALEN endonucleases that cleave within the AAVS1 locus. Three weeks after transfection, a distinct 7% subpopulation of cells expressed IL2RG on the cell surface, which was subsequently sorted by flow cytometry. We analyzed 10 single cell subclones from this population and confirmed that homologous recombination and single allele targeting had occurred in each of these clones, demonstrating efficient homologous recombination at the AAVS1 site. Flow analysis showed that the EF1α-IL2RG-cDNA cassette was expressed at approximately the same level as that seen in cells transduced with a single copy of the clinical EF1α-IL2RG-cDNA lentiviral vector, verifying that the AAVS1 is permissive for therapeutic expression levels at the single copy level. One potential disadvantage of the safe harbor approach is the potential loss of precise regulation of gene expression resulting from the use of such synthetic cDNA constructs. For this reason, we are also currently targeting the full 5.3 kb genomic IL2RG locus and endogenous promoter to the AAVS1 safe harbor to compare their IL2RG expression level with that obtained from the cDNA construct. We are also comparing editing efficiency at AAVS1 using several TALEN pairs as well as 4 Staphylococcus Aureus Cas9 guide RNAs, which are deliverable by “all-in-one” single stranded AAV6 vectors. To date, the best guide has led to 37 % allele editing in 293T cells when transfected as plasmid. We are currently producing AAV vectors that contain this guide RNA and will test this AAV vector, along with IDLV vectors that will transfer either the cDNA or genomic IL2RG templates, for editing efficiency and IL2RG gene expression in ED7R and human CD34+ cells.

398. Site-Specific Introduction of Chimeric Antigen Receptors to Primary Human T Cells

In current clinical trials, delivery of an anti-cancer chimeric antigen receptor (CAR) to patient T cells is accomplished using randomly integrating retroviruses. Designer nucleases and AAV donor templates allow an alternative delivery method that introduces the CAR gene cassette at a target locus via homologous recombination, with concomitant disruption of the endogenous gene. Here we show high-efficiency introduction of anti-CD19 and anti-BCMA CAR expression cassettes in primary human T cells at two clinically relevant loci: CCR5 and TCRa. These gene-targeted CAR+ cells (tCARs) exhibit equivalent activity in vitro against CD19+ and BCMA+ cell lines compared to lentiviral-delivered CARs, and effectively clear tumor in a murine xenograft model. Advantages over lentiviral delivery include utilization of a defined integration site for the CAR construct linked with endogenous gene disruption. The use of specific nucleases and AAV for CAR delivery expands the possibilities for a precisely engineered cell therapy product in the clinic. For example, a CCR5-tCAR may be clinically useful in HIV+ patients, where traditional delivery methods leave therapeutic cells vulnerable to HIV infection and elimination. Moreover, a TCRa-tCAR could allow for off-the-shelf CAR therapy, potentially removing limitations to current approaches. Finally, the approach described here is broadly applicable for targeted delivery of alternative therapeutic cassettes at translationally relevant sites across the human genome.

Efficient production of superior dumbbell-shaped DNA minimal vectors for small hairpin RNA expression.

Genetic therapy holds great promise for the treatment of inherited or acquired genetic diseases; however, its breakthrough is hampered by the lack of suitable gene delivery systems. Dumbbell-shaped DNA minimal vectors represent an attractive, safe alternative to the commonly used viral vectors which are fraught with risk, but dumbbell generation appears to be costly and time-consuming. We developed a new PCR-based method for dumbbell production which comprises only two steps. First, PCR amplification of the therapeutic expression cassette using chemically modified primers to form a ready-to-ligate DNA structure; and second, a highly efficient intramolecular ligation reaction. Compared with conventional strategies, the new method produces dumbbell vectors more rapidly, with higher yields and purity, and at lower costs. In addition, such produced small hairpin RNA expressing dumbbells triggered superior target gene knockdown compared with conventionally produced dumbbells or plasmids. Our novel method is suitable for large-scale dumbbell production and can facilitate clinical applications of this vector system.

Therapeutic platform for immunotherapy of Chagas disease (VAC9P.1106)

Abstract Chagas Disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is a leading cause of heart disease and sudden cardiac death in Latin America, disproportionately affecting people living in resource-poor areas. Approved treatment is limited to two drugs, benznidazole and nifurtimox, which have poor efficacy in the chronic stage of disease as well as severe side effects. Thus, there is an urgent need for alternative therapies to treat Chagas Disease. Pre-clinical models have demonstrated that antigen specific CD8+IFNγ+ responses are essential for reducing parasite burdens, increasing survival, and decreasing cardiac pathology in both the acute and chronic phases of Chagas. In the present study, we developed an adenovirus-based gene transfer cassette that was used therapeutically to prevent or delay the cardiac complications that result from infection with T. cruzi. The therapeutic cassette, containing T. cruzi Tc24 and TSA1 antigens and a dominant-negative isoform of the SHP-1 inhibitory phosphatase, mediated development of antigen-specific CD8+IFNγ+ cells when ex vivo-transduced dendritic cells were administered to Balb/c mice. Additionally, the vaccine platform reduced parasite burdens, increased survival, and reduced cardiac pathology in a Balb/c model of live T. cruzi infection. The data suggest that immunotherapeutic approaches might be successful in addressing the unmet medical need for improved Chagas treatments.

Self-illuminative cascade-reaction-driven anticancer therapeutic cassettes made of cooperatively interactive nanocomplexes

Therapeutic options based on near-infrared (NIR) wavelengths have attracted attention owing to in vivo lowest-background interventions and the development of several nano-architectures with localized surface plasmon resonance. Because of their limited tissue penetration, the clinical use of NIR light-driven treatments is not widespread; this technology is inapplicable to infection sites in the deeper areas of internal tissues. In this study, we demonstrate a self-illuminative therapeutic cassette able to exert anticancer effects via a series of enzymatic, chemical, and optical cooperative cascade reactions. It consists of (1) NIR-illuminative nanocomplexes and (2) NIR-sensitive therapeutic cassettes, which demonstrate a 60% chemically-induced killing effect in a prostate cancer model without external NIR irradiation. This technology can also be actively exploited as an imaging agent due to adaptation of a self-illuminating nanocomplex. Consequently, these novel therapeutic cassettes, which work not only as a powerful internal NIR stimulant, but also as a biological imaging platform, provide a new rational design concept for biomedical use.

Rational Design for Enhanced Gene Therapy with DNA Transposons

Several genetic diseases caused by deficiencies in enzymes required throughout the body can be treated by enzyme replacement therapy at costs of upward of hundreds of thousands of dollars per year per patient. In the long run, such costs are prohibitive. Gene therapy is therefore an appealing option in that its aim is to treat patients after only a single or very few interventions. Clinical-grade viral vectors can efficiently deliver therapeutic expression cassettes, but such viruses can still be expensive to prepare. By contrast, clinical-grade nonviral nucleic acids are relatively inexpensive to produce but difficult to deliver into cells. In this issue of Molecular Therapy, two reports from the same group describe improvements to two limiting factors of nonviral gene therapy using DNA transposons delivered to the liver.1,2 The authors substantially increased efficiency of delivery of transgenes to the target organ and the amount of therapeutic protein exported per cell following transgene delivery.

Toward a Safer Integration Profile of MLV-based Retroviral Vectors

Toward a Safer Integration Profile of MLV-based Retroviral Vectors

A Light‐Driven Anti‐Cancer Dual‐Therapeutic Cassette Enhances Solid Tumour Regression

The majority of anticancer therapeutics have failed to control the target cancers. Thus, new rational design concepts are critical. In most of the biological reactions, a cascade pathway is used to activate appropriate responses. In the cascade pathway, a small signal derived from neighboring environments can be amplified and it further triggers overwhelming and specialized responses. It can be applied to achieve powerful therapeutic effects for novel drug design strategies. Inspired by this concept, we design a preferential dual anti-cancer therapeutic cassette composed of (i) DNA/RNA nanostructures as both anticancer containers and target ligands and (ii) a gold nanocrystal as localized heat inducers. We demonstrate that this multi-modular platform is superior to conventional cancer medications in that it had higher drug loading efficiency, tunable drug release, and intrinsic serum stability characteristics. Both doxorubicin chemotherapy and thermal ablation exert a powerful synergistic killing effect that resulted in prostate cancer regression both in vitro and in vivo. We speculate that our novel anti-cancer drug system can be adapted to effectively destroy many different types of solid cancers.

Safety profile, efficacy, and biodistribution of a bicistronic high-capacity adenovirus vector encoding a combined immunostimulation and cytotoxic gene therapy as a prelude to a phase I clinical trial for glioblastoma

Adenoviral vectors (Ads) are promising gene delivery vehicles due to their high transduction efficiency; however, their clinical usefulness has been hampered by their immunogenicity and the presence of anti-Ad immunity in humans. We reported the efficacy of a gene therapy approach for glioma consisting of intratumoral injection of Ads encoding conditionally cytotoxic herpes simplex type 1 thymidine kinase (Ad-TK) and the immunostimulatory cytokine fms-like tyrosine kinase ligand 3 (Ad-Flt3L).Herein, we report the biodistribution, efficacy, and neurological and systemic effects of a bicistronic high-capacity Ad, i.e., HC-Ad-TK/TetOn-Flt3L. HC-Ads elicit sustained transgene expression, even in the presence of anti-Ad immunity, and can encode large therapeutic cassettes, including regulatory elements to enable turning gene expression “on” or “off” according to clinical need. The inclusion of two therapeutic transgenes within a single vector enables a reduction of the total vector load without adversely impacting efficacy. Because clinically the vectors will be delivered into the surgical cavity, normal regions of the brain parenchyma are likely to be transduced. Thus, we assessed any potential toxicities elicited by escalating doses of HC-Ad-TK/TetOn-Flt3L (1×108, 1×109, or 1×1010 viral particles [vp]) delivered into the rat brain parenchyma. We assessed neuropathology, biodistribution, transgene expression, systemic toxicity, and behavioral impact at acute and chronic time points. The results indicate that doses up to 1×109 vp of HC-Ad-TK/TetOn-Flt3L can be safely delivered into the normal rat brain and underpin further developments for its implementation in a phase I clinical trial for glioma.

Radiolabeled Nucleoside Analogues for PET Imaging of HSV1-tk Gene Expression

The HSV1-tk gene has been explored as a reporter and/or suicide gene in molecular imaging of gene expression. Gene therapy with HSV1-tk and the use of this gene as a marker have been applied in patients with various forms of cancer. However, the conditions for clinical gene therapy protocols have yet to be optimized. A method to monitor the activity of HSV-tk in vivo would be extremely useful to optimize clinical gene therapy protocols. Positron emission tomography (PET) with a suitable probe can offer information about both the extent of gene expression and its distribution, provided that an appropriate reporter gene is included in the therapeutic cassette. PET imaging provides higher resolution and sensitivity and allows noninvasive quantification of biological processes. Several radiolabeled pyrimidine (thymidine) and purine (acycloguanosine) derivatives have been developed as reporter probes for imaging of HSV-TK enzyme activity with PET. In this review, information on radiolabeling and PET imaging of HSV1-tk gene expression with various nucleoside analogues is presented.

Preclinical Studies for Gene Therapy of Duchenne Muscular Dystrophy

The muscular dystrophies are a diverse group of genetic disorders without an effective treatment. Because they are caused by mutations in various genes, the most direct way to treat them involves correcting the underlying gene defect (ie, gene therapy). Such a gene therapy approach involves delivering a therapeutic gene cassette to essentially all the muscles of the body in a safe and efficacious manner. The authors describe gene delivery methods using vectors derived from adeno-associated virus that are showing great promise in preclinical studies for treatment of Duchenne muscular dystrophy. It is hoped that variations on these methods might be applicable for most, if not all, of the different types of muscular dystrophy.