Transgene Expression Research Articles

Abstract 7248: Simultaneous isolation and parallel analysis of genomic DNA and RNA for gene therapy

Abstract Introduction Viral vectors, such as adeno-associated vector (AAV), play a crucial role in delivering transgenes for gene therapy research. The success of vector transduction is vital for efficacy, which is measured through vector copy number (VCN) and transgene expression. In preclinical studies, both VCN and transgene expression are commonly tested from the same sample to ensure accuracy. We have reported a method for simultaneous high-quality genomic DNA and RNA extraction without splitting the sample, utilizing SPRI bead-based technology that has consistent performance across diverse tissue types and brain regions. Methods AAV was administered to mouse and non-human primate (NHP) models. At the conclusion of each experiment, tissue samples from the adrenal gland, brain, gonads, heart, kidney, liver, lung, skeletal muscle, spinal cord, and spleen were harvested. All tissue samples that demonstrated around 15 mm3 were snap-frozen in liquid nitrogen before nucleic acid extraction. The extraction process was carried out manually or on an automated liquid handler. The quality and quantity of DNA and RNA were then assessed from all tested tissues before downstream assay. Results Our study found that all mouse tissue types (n=72/tissue type) and NHP (n=11/tissue type) had an average gDNA and RNA yield that exceeded the minimum yield cutoff of 25 ng/μL for DNA and 6.25 ng/μL for RNA. We also analyzed DNA from tissues treated with two different AAV serotype-treated tissues in the mouse model, which showed that transduction across all the tissues was highly efficient for both AAV serotypes. Furthermore, we observed variations in transgene expression levels (copies/1 μg RNA) among different tissue types. Similar patterns of VCN and transgene expression were also observed in NHP models. Conclusions Efficient gene therapy depends on a safe and effective viral vector. The simultaneous extraction of gDNA and RNA simplifies the process, saves time, ensures quality, and facilitates integration into existing lab automation. This leads to consistent and error-resistant performance in analyzing vector copy and transgene levels. Citation Format: Han Wei. Simultaneous isolation and parallel analysis of genomic DNA and RNA for gene therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7248.

Abstract 2182: 15-lipoxygenase-1 modulation of colonic resolvin production to suppress colorectal carcinogenesis

Abstract Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are widely used dietary supplements and FDA-approved treatments. The reported impacts of fish oil, DHA, and EPA on colorectal carcinogenesis (CRC) have spurred debates, as certain studies suggest promotional effects while numerous others emphasize suppressive effects. Nevertheless, resolvins, oxidative metabolites of EPA and DHA, exhibit the ability to inhibit crucial pathways (such as TNF-α and IL-1β) that contribute to CRC. 15-lipoxygenase-1 (ALOX15) plays a crucial role in the oxidative metabolism of DHA and EPA, ultimately leading to the formation of resolvins. However, ALOX15 expression is commonly lost during human CRC. Whether loss of ALOX15 expression modulates the effects of DHA and EPA on CRC remains to be elucidated. We therefore conducted the experiments in preclinical CRC mouse models induced by Azoxymethane or Apc gene mutation to explore the hypothesis that ALOX15 expression in colonic epithelial cells regulates DHA and EPA metabolism to generate resolvins, subsequently influencing CRC outcomes. We found that 1) DHA and EPA exert differential effects on CRC, which can be further modified by factors such as their formulation (e.g., ethyl ester versus triglyceride); 2) targeted transgenic expression of ALOX15 in the intestines of mice (referred to as ALOX15-Gut mice) markedly augmented the production of resolvins and concurrently suppressed CRC; 3) the predominant products from the expressed ALOX15 enzymatic activity were identified as 17-HDHA and resolvin D5; and 4) the effects ALOX15 on increasing resolvin generation and suppressing CRC were consistent across various promoters used to drive ALOX15 transgenic expression in mice. These findings collectively underscore the profound importance of ALOX15 intestinal expression as a host-related factor in resolvin generation from EPA and DHA, which subsequently contributes to the suppression of CRC. Citation Format: Xiangsheng Zuo, Yoshiyuki Kiyasu, Yi Liu, Micheline Moussalli, Bo Wei, Peiying Yang, Imad Shureiqi. 15-lipoxygenase-1 modulation of colonic resolvin production to suppress colorectal carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2182.

Abstract 3995: A novel chimeric Fas signal redirect receptor enhances the durability of anti-tumor activity and serial killing potential of CAR T cells

Abstract Chimeric antigen receptor (CAR) T-cell therapies have demonstrated remarkable success in hematological malignancies but only modest efficacy in solid tumors. Apoptosis induced by Fas receptor signaling can be a substantial barrier to CAR T-cell antitumor activity, as many solid tumors express high levels of Fas ligand (FasL). Here we describe a novel synthetic construct where the ectodomain of Fas receptor is fused to the signaling endodomain of a stimulatory receptor, creating a Fas signal redirect receptor (Fas-SRR). This unique design not only prevents Fas-dependent cell death, but also augments the long-term killing potential of induced pluripotent stem cell (iPSC)-derived CAR-T (CAR-iT) cells. Fas-SRR constructs with unique T-cell activation signaling endodomains were designed and compared to two control arms: a dominant negative receptor (Fas-DNR), which blocks Fas signal but does not promote activation; and a Fas-41BB fusion receptor, where the 41BB endodomain is fused to FasR ectodomain to direct activation upon FasL:Fas engagement. Fas-SRR designs were validated in Jurkat reporter cells treated with Fas agonist to measure signal redirection via Fas-SRR. To conduct a head-to-head comparison, CAR-iT cells were engineered to express a CAR under the regulation of the TRAC locus and to express either a Fas-DNR, a Fas-41BB, or FAS-SRR constructs at high purity (>90% transgene expression). No major differences were seen in maturation phenotype (>95% TRAC-CAR expression) of these cells, indicating compatibility of the FAS-SRR constructs with CAR-iT cell generation. Functional testing of the Fas-SRR+ CAR-iT cells was performed in a long-term restimulation assay where effector cells were repeatedly exposed to target cells over multiple rounds of co-culture. Complete loss of target cell lysis was observed by round six in the co-cultures with FAS-DNR cells, where loss of CAR-iT cell persistence led to accumulation of target cells. After eight rounds of co-culture, the Fas-41BB cells and the majority of Fas-SSR cell constructs also began to exhibit minimal activity toward target cells. However, the top Fas-SRR cell constructs continued to show durable antitumor activity (>97% cytolysis) through the end of the 9-round rechallenge assay. Of note, GSEA analysis identified significant upregulation of NFkB and RELA target genes in cells having the top FAS-SRR construct versus the less durable designs. These results reinforce the advantage of attenuating Fas-FasL interactions to prevent apoptosis and, more importantly, demonstrate the unique therapeutic value in redirecting Fas signaling to improve T-cell potency over an extended duration. Additional studies are underway to further investigate the implications of Fas signal redirection in CAR T and CAR-iT cells and to further understand the module of genes involved in supporting serial stimulation performance. Citation Format: Christopher Ecker, Eigen Peralta, Hui-Yi Chu, Lorraine Loter, Masanao Tsuda, Earl Avramis, Matthew Denholtz, Alec Witty, Tom Lee, Bahram Valamehr. A novel chimeric Fas signal redirect receptor enhances the durability of anti-tumor activity and serial killing potential of CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3995.

Abstract 772: Investigation of gender disparity in liver tumor formation using a hydrodynamic tail vein injection mouse model

Abstract The incidence of hepatocellular carcinoma (HCC), the major form of liver cancer, is 4 times higher in men than in women. This gender disparity is difficult to study since it is believed to be multifactorial, due in part to differences in risk factors, behaviors, and hormones. The liver is a sexually dimorphic organ that is extremely susceptible to interactions with estrogens and androgens. Estrogen has been proven to have a protective effect against HCC with a demonstrable gender-related hormonal effect on the growth of HCC, though the underlying mechanisms are not fully understood. The hydrodynamic tail vein injection (HTVI) mouse model allows for the induction of liver tumors in the host. Eliminating the social and behavioral aspects of the gender disparity will allow us to determine what role estrogen plays in tumor development. In our study, a gender disparity in tumor development was observed between 6-week-old male and female FVB/NJ mice injected via HTVI of plasmids expressing c-MET, a mutated beta-catenin (b-Cat) and Sleeping Beauty transposon. HTVI of a Luciferase and Sleeping Beauty transposon expressing plasmid showed no significant difference in photon flux by bioluminescence imaging between male and female mice 24 hours after injection. When imaging was repeated one week after injection, the photon flux reduced by an order of magnitude in both male and female mice with the female mice showing more uniform reduction than the male mice. Similar luciferase gene copy number was detected in the liver tissue of male and female mice. These data suggest that there appears to be no gender difference in the transient expression and stable integration of the transfected plasmid. Plasma alpha-fetoprotein (AFP) was used to monitor tumor formation in the mice that were injected with c-MET and b-Cat without luciferase as luciferase is immunogenic. The male mice had significantly higher AFP levels at 3.5 weeks, 8 weeks, and 12 weeks post-injection than the female mice. At termination, the liver weight confirmed that female mice had much lower tumor burden than the males. We are currently using quantitative PCR to confirm similar copy number of exogenous c-MET and b-Cat inserted in the tumor cell genome and using RT-qPCR and Western blotting to determine whether the transgene expression is different in tumors from male and female mice. To determine whether estrogen played a role in preventing tumor development and progression in female mice, six-week-old male and ovariectomized female mice have been injected with the same oncogene plasmid combination plus the sleeping beauty. Interestingly, AFP levels between male and ovariectomized female mice were comparable when measured at 3 weeks. We will present our findings at the meeting. Citation Format: Araceli Bernal, Morgana McLaughlin, Ankur Tiwari, Francisco Cigarroa, LuZhe Sun. Investigation of gender disparity in liver tumor formation using a hydrodynamic tail vein injection mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 772.

Abstract 7132: Preclinical characterization of LAE119, a novel PARP1 selective inhibitor and trapper

Abstract Current PARP inhibitors primarily target PARP1/2 and demonstrate high efficacy against tumors with deficiencies in homologous recombination repair, such as those with BRCA mutations. However, these inhibitors are also associated with significant hematological toxicities. Studies have indicated that the synthetic lethality observed with BRCA mutations is mainly driven by PARP1, while PARP2 is not essential in this context. PARP2 has been shown to be crucial for the survival of hematopoietic stem/progenitor cells in animal models. Therefore, selective inhibition of PARP1 could potentially reduce toxicity and improve therapeutic outcomes. Here we introduce LAE119, a potent and selective PARP1 inhibitor and PARP1-DNA trapper. It demonstrates more than a 1000-fold selectivity for PARP1 DNA trapping compared to PARP2. In comparison to most PARP inhibitors including AZD5305, LAE119 exhibits extremely long residence time on PARP1 in both biochemical and cellular assays. It demonstrates robust anti-proliferation activity against BRCA2−/− DLD-1 cells and MDA-MB-436 cells, with IC50 value of 0.6 nM in both cell lines. In CAL51 cells, which has a BRCA2 mutation but with low PARP1 expression level and are much less sensitive to AZD5305, LAE119 demonstrates much better activity than AZD5305, particularly when PARP1 level is increased with transgene expression. LAE119 demonstrated substantial tumor inhibition in MDA-MB-436 and Capan-1 xenograft models. LAE119 also displays favorable ADME properties and PK profiles. It has high tumor/plasma ratio which is beneficial for sustainable anti-tumor effect and reduction of off-target effects. In rat hematologic toxicity study, LAE119 at 10 mpk QD dose was well tolerated and had minimal effects on hematologic parameters. Both in vitro and in vivo data strongly support the further clinical investigation of LAE119. Citation Format: Ming Li, Yan Chen, Junyan Chen, Ling Jiang, Xiaofen Lin, Justin Gu. Preclinical characterization of LAE119, a novel PARP1 selective inhibitor and trapper [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7132.

Abstract 6328: Preclinical development of CAR T cells with antigen-inducible IL-18 enforcement to treat GD2-positive solid cancers

Abstract Cytokine-engineering is a promising strategy to overcome limitations of CAR T cell therapy in solid tumors. We have developed a novel investigational medicinal product, consisting of CAR T cells directed against ganglioside GD2 along with CAR-inducible secretion of IL-18, to enforce their activation response and effector functions in the microenvironment of solid cancers. Our lentiviral all-in-one vector combines constitutive expression of a 14.G2a antibody-derived GD2-specific, 4-1BB-costimulated CAR with inducible nuclear factor of activated T cells (NFAT)-driven expression of the human IL-18 transgene. A control construct contains EGFP in the place of IL-18 (GD2EGFPCART). Lentiviral transduction of T cells from various individual donors with both preclinical and clinical batches of virus resulted in CAR surface expression in >40% of T cells. GD2IL18CART contained effector memory and central memory T cell phenotypes at similar frequencies as the control. CAR engagement by immobilized anti-idiotype antibody or by GD2-positive tumor cells selectively induced IL-18 release, with production of IL-18 strictly depending on GD2 antigen engagement. GD2IL18CART responded to interaction with GD2-positive tumor cells with superior IFN-γ, TNF-α and IL-2 cytokine release and superior target cytolysis compared to the control CAR T cells without inducible IL-18, whereas IL-6 was secreted at similar concentrations. In an NSG xenograft model of disseminated GD2-positive tumors, GD2IL18CART had superior in vivo anti-tumor activity than GD2EGFPCART, with eradication of GD2-positive tumor xenografts as detected by bioluminescence imaging while the control achieved only limited extension of progression-free survival. Finally, we established GMP-compliant manufacturing of GD2IL18CART that is feasible and efficient at clinical scale, using an automated protocol and device. Three validation runs confirmed robust expansion of CAR+ T cells to final cell numbers of 3.5x109 CAR+ cells (median; range 3.4 to 3.7x109), with consistent transduction efficiencies of >54% at the in-process control time-point and at harvest. GD2IL18CART from clinical-scale manufacturing runs responded to interaction with GD2 with strictly antigen-dependent secretion of both IL-18 and IFN-γ and efficiently lysed various GD2-positive tumor targets. In conclusion, we assembled all prerequisites for clinical evaluation of an IL-18 cytokine enhanced, GD2-specific CAR T cell product for the treatment of patients with a variety of solid tumors, recruited on the basis of GD2 immunofluorescence pre-screening (EU CT 2022-501725-21-00). Besides addressing the safety of GD2IL18CART therapy in pediatric and adult patients as the primary endpoint, the study will allow to gain substantial knowledge on the biological effects of IL-18 in the TME of the individual solid tumors. Citation Format: Lena Fischer-Riepe, Sareetha Kailayangiri, Katharina Zimmermann, Rita Pfeifer, Michael Aigner, Bianca Altvater, Sascha Kretschmann, Simon Voelkl, Jordan Hartley, Celine Dreger, Katja Petry, Andreas Bosio, Angelika von Doellen, Wolfgang Hartmann, Holger Lode, Dennis Goerlich, Andreas Mackensen, Melanie Jungblut, Axel Schambach, Hinrich Abken, Claudia Rossig. Preclinical development of CAR T cells with antigen-inducible IL-18 enforcement to treat GD2-positive solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6328.

Abstract 4012: SOT302: Dual expression of exogenous glutamine oxaloacetate transaminase (GOT2) and TP53-induced glycolysis and apoptosis regulator (TIGAR) enhance CAR T cell activity in preclinical solid tumor models

Abstract Background: Despite success in hematological malignancies, CAR T therapy has had limited efficacy against solid tumors. The suppressive tumor microenvironment limits CAR T activity through various mechanisms including competition for available nutrients and chronic stimulation resulting in reduced effector functions or T cell exhaustion. CAR T cells with enhanced metabolic fitness or more durable memory phenotype could potentially improve the clinical outcome in solid tumors. GOT2 plays an important role in mitochondrial function and maintenance of redox homeostasis. We have previously demonstrated enhancement of CAR T cells overexpressing GOT2. TIGAR is an enzyme known to promote antioxidative activites and reduce reactive oxygen species and has a role in protecting against apoptosis. Here we tested SOT302 CAR T cells expressing both GOT2 and TIGAR in preclinical solid tumor models. Results: Expression of GOT2 and TIGAR transgenes were confirmed by qRT-PCR and western blot. Activity of GOT2 and TIGAR were confirmed by measuring aminotransferase activity (AST) and glutathione production, respectively. Compared to control CAR T cells, SOT302 is enriched for CD8+ Tscm cells and has a higher percentage functional, non-senescent (CD28+CD57-) cells. SOT302 has similar cytokine expression and cytotoxicity in standard co-culture assays compared to controls. SOT302 cells were chronically stimulated with plate-bound antigen every 3-4 days for 4 rounds. SOT302 had greater expansion of cells and were less exhausted and had better cytolytic capacity compared to controls. SOT302 cells were then evaluated in tumor xenograft mouse models. Consistent with our previous findings, CAR T cells expressing the single GOT2 transgene were more efficacious compared to CAR T cells alone. Expression of the single TIGAR transgene had no apparent benefit over CAR alone, however SOT302 cells expressing both GOT2 and TIGAR had superior anti-tumor activity compared to CAR alone and CAR+GOT2. No overt toxicity or significant body weight loss was observed. Ex vivo analyses to measure peripheral expansion, tumor infiltration and exhaustion were also performed. Preliminary findings indicate that SOT302 has better peripheral expansion compared to CAR alone and similar expansion compared to CAR+GOT2 controls. SOT302 cells had better tumor infiltration compared to both the CAR alone and CAR+GOT2 cells. Tumor infiltrating SOT302 cells also exhibited lower levels of exhaustion compared to CAR alone or CAR+GOT2 controls. Summary: SOT302 cells express exogenous GOT2 and TIGAR transgenes and have superior anti-tumor activity in preclinical solid tumor models. These data suggest that SOT302 may be a promising candidate for patients with solid tumor cancers. Citation Format: Emily Kuiper, Samyabrata Bhaduri, Daniel Garafola, Kshitij Sharma, Jennifer Coccia, Kathleen Whiteman, Amy Jensen-Smith. SOT302: Dual expression of exogenous glutamine oxaloacetate transaminase (GOT2) and TP53-induced glycolysis and apoptosis regulator (TIGAR) enhance CAR T cell activity in preclinical solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4012.

Reprograming skin fibroblasts into Sertoli cells: a patient-specific tool to understand effects of genetic variants on gonadal development

BackgroundDisorders/differences of sex development (DSD) are congenital conditions in which the development of chromosomal, gonadal, or anatomical sex is atypical. With overlapping phenotypes and multiple genes involved, poor diagnostic yields are achieved for many of these conditions. The current DSD diagnostic regimen can be augmented by investigating transcriptome/proteome in vivo, but it is hampered by the unavailability of affected gonadal tissue at the relevant developmental stage. We try to mitigate this limitation by reprogramming readily available skin tissue-derived dermal fibroblasts into Sertoli cells (SC), which could then be deployed for different diagnostic strategies. SCs form the target cell type of choice because they act like an organizing center of embryonic gonadal development and many DSD arise when these developmental processes go awry.MethodsWe employed a computational predictive algorithm for cell conversions called Mogrify to predict the transcription factors (TFs) required for direct reprogramming of human dermal fibroblasts into SCs. We established trans-differentiation culture conditions where stable transgenic expression of these TFs was achieved in 46, XY adult dermal fibroblasts using lentiviral vectors. The resulting Sertoli like cells (SLCs) were validated for SC phenotype using several approaches.ResultsSLCs exhibited Sertoli-like morphological and cellular properties as revealed by morphometry and xCelligence cell behavior assays. They also showed Sertoli-specific expression of molecular markers such as SOX9, PTGDS, BMP4, or DMRT1 as revealed by IF imaging, RNAseq and qPCR. The SLC transcriptome shared about two thirds of its differentially expressed genes with a human adult SC transcriptome and expressed markers typical of embryonic SCs. Notably, SLCs lacked expression of most markers of other gonadal cell types such as Leydig, germ, peritubular myoid or granulosa cells.ConclusionsThe trans-differentiation method was applied to a variety of commercially available 46, XY fibroblasts derived from patients with DSD and to a 46, XX cell line. The DSD SLCs displayed altered levels of trans-differentiation in comparison to normal 46, XY-derived SLCs, thus showcasing the robustness of this new trans-differentiation model. Future applications could include using the SLCs to improve definitive diagnosis of DSD in patients with variants of unknown significance.

Genome-wide identification and characterization of xyloglucan endotransglucosylase/hydrolase gene family in maize (Zea mays L.) and the function of ZmXTH30 in response to drought stress

The xyloglucan endotransglucosylase/hydrolase (XET/XEH, also known as XTH) family is a crucial multigene family in plants, playing a key role in cell wall reconstruction and stress tolerance. While the ZmXTH genes in maize (Zea mays L.) have not been thoroughly studied, this research aimed to identify and characterize them on a genome-wide scale. A total of 31 ZmXTH genes were identified, with phylogenetic analyses indicating their closest relation to rice (Oryza sativa L.) OsXTH family members. Evolutionary analysis revealed genetic variation in ZmXTH genes during the shift from dicotyledons to monocotyledons, with evidence of purifying selection throughout their evolution. Notably, ZmXTH14, ZmXTH26, and ZmXTH30 showed significant upregulation under abiotic stress, suggesting their importance in stress responses. Furthermore, transgenic expression of ZmXTH30 in Arabidopsis (Arabidopsis thaliana) improved seedling growth under drought stress by reducing reactive oxygen species and enhancing antioxidant enzyme activity, underscoring the role of ZmXTH30 in plant drought tolerance. These findings provide valuable insights for further exploring the functions and applications of ZmXTH genes in maize.

The presence of CpGs in AAV gene therapy vectors induces a plasmacytoid dendritic cell-like population very early after administration

AAV-mediated gene transfer is a promising platform still plagued by potential host-derived, antagonistic immune responses to therapeutic components. CpG-mediated TLR9 stimulation activates innate immune cells and leads to cognate T cell activation and suppression of transgene expression. Here, we demonstrate that CpG depletion increased expression of an antibody transgene product by 2–3-fold as early as 24 h post-vector administration in mice. No significant differences were noted in anti-transgene product/ anti-AAV capsid antibody production or cytotoxic gene induction. Instead, CpG depletion significantly reduced the presence of a pDC-like myeloid cell population, which was able to directly bind the antibody transgene product via Fc-FcγR interactions. Thus, we extend the mechanisms of TLR9-mediated antagonism of transgene expression in AAV gene therapy to include the actions of a previously unreported pDC-like cell population.

Physiologically based modeling of LNP-mediated delivery of mRNA in the vascular system

RNA therapeutics are an emerging, powerful class of drugs with potential applications in a wide range of disorders. A central challenge in their development is the lack of clear pharmacokinetic-pharmacodynamic relationship, in part due to the significant delay between the kinetics of RNA delivery and the onset of pharmacologic response. To bridge this gap, we have developed a physiologically-based pharmacokinetic/pharmacodynamic model for systemically administered mRNA-containing lipid nanoparticles in mice. This model accounts for the physiologic determinants of mRNA delivery, active targeting in the vasculature, and differential transgene expression based on nanoparticle coating. The model was able to well-characterize the blood and tissue pharmacokinetics of lipid nanoparticles, as well as the kinetics of tissue luciferase expression measured by ex vivo activity in organ homogenates and bioluminescence imaging in intact organs. The predictive capabilities of the model were validated using a formulation targeted to intercellular adhesion molecule-1 and the model predicted nanoparticle delivery and luciferase expression within 2-fold error for all organs. This modeling platform represents an initial strategy that can be expanded upon and utilized to predict the in vivo behavior of RNA-containing lipid nanoparticles developed for an array of conditions and across species.

CD40 stimulation via CD40 ligand enhances adenovirus-mediated tumour immunogenicity including 'find-me', 'eat-me', and 'kill-me' signalling.

Immunostimulatory gene therapy using oncolytic viruses is currently evaluated as a promising therapy for cancer aiming to induce anti-tumour immunity. Here, we investigate the capacity of oncolytic adenoviruses (LOAd) and their transgenes to induce immunogenicity in the infected tumour cells. Oncolysis and death-related markers were assessed after infection of eight human solid cancer cell lines with different LOAd viruses expressing a trimerized, membrane-bound (TMZ)-CD40L, TMZ-CD40L and 41BBL, or no transgenes. The viruses induced transgene expression post infection before they were killed by oncolysis. Death receptors TRAIL-R1, TRAIL-R2 and Fas as well as immunogenic cell death marker calreticulin were upregulated in cell lines post infection. Similarly, caspase 3/7 activity was increased in most cell lines. Interestingly, in CD40+ cell lines there was a significant effect of the TMZ-CD40L-encoding viruses indicating activation of the CD40-mediated apoptosis pathway. Further, these cell lines showed a significant increase of calreticulin, and TRAIL receptor 1 and 2 post infection. However, LOAd viruses induced PD-L1 upregulation which may hamper anti-tumour immune responses. In conclusion, LOAd infection increased the immunogenicity of infected tumour cells and this was potentiated by CD40 stimulation. Due to the simultaneous PD-L1 increase, LOAd viruses may benefit from combination with antibodies blocking PD1/PD-L1.

Harnessing whole human liver ex situ normothermic perfusion for preclinical AAV vector evaluation

Developing clinically predictive model systems for evaluating gene transfer and gene editing technologies has become increasingly important in the era of personalized medicine. Liver-directed gene therapies present a unique challenge due to the complexity of the human liver. In this work, we describe the application of whole human liver explants in an ex situ normothermic perfusion system to evaluate a set of fourteen natural and bioengineered adeno-associated viral (AAV) vectors directly in human liver, in the presence and absence of neutralizing human sera. Under non-neutralizing conditions, the recently developed AAV variants, AAV-SYD12 and AAV-LK03, emerged as the most functional variants in terms of cellular uptake and transgene expression. However, when assessed in the presence of human plasma containing anti-AAV neutralizing antibodies (NAbs), vectors of human origin, specifically those derived from AAV2/AAV3b, were extensively neutralized, whereas AAV8- derived variants performed efficiently. This study demonstrates the potential of using normothermic liver perfusion as a model for early-stage testing of liver-focused gene therapies. The results offer preliminary insights that could help inform the development of more effective translational strategies.

Generation of an enhancer-driven gene expression viral tool specific to dentate granule cell-types through direct hippocampal injection.

Accurate investigations of neural circuitry require specific genetic access to individual circuit elements, i.e., the myriad neuronal cell-types in the brain. However, native promoters cannot achieve this because while most genes are expressed in the brain, few are expressed in a single neuronal cell-type. We recently used enhancers, the subcomponents of the transcriptional apparatus which tell promoters when and where to express, combined with heterologous minimal promoters to increase specificity of transgene expression, an approach we call Enhancer-Driven Gene Expression (EDGE). As we discuss, EDGE is a marked improvement in specificity over native promoters, but still requires careful anatomical analysis to avoid off-target effects. In this study we present a more complete set of genomic markers from the mouse brain and characterize a novel EDGE viral vector capable of specifically driving expression in distinct subtypes of hippocampal neurons, even though it can express in other cell-types elsewhere. The advent of cell-type specific viral tools in wild-type animals provides a powerful strategy for neural circuit investigation and holds promise for studies using animal models for which transgenic tools are not available.

Adeno-associated virus-mediated trastuzumab delivery to the central nervous system for human epidermal growth factor receptor 2+ brain metastasis.

Trastuzumab improves overall survival for HER2+ breast cancer, but its short half-life in the cerebrospinal fluid (~2-4 days) and delivery limitations restrict the ability to target HER2+ central nervous system (CNS) disease. We developed an adeno-associated virus (AAV) vector expressing a codon-optimized, ubiquitin C (UbC)-promoter-driven trastuzumab sequence (AAV9.UbC.trastuzumab) for intrathecal administration. Transgene expression was evaluated in adult Rag1 knockout mice and rhesus nonhuman primates (NHPs) after a single intracerebroventricular (ICV) or intra-cisterna magna (ICM) AAV9.UbC.trastuzumab injection, respectively, using real-time PCR, ELISA, Western blot, in situ hybridization, single-nucleus RNA sequencing, and liquid chromatography-mass spectrometry; antitumor efficacy was evaluated in brain xenografts using HER2+ breast cancer cell lines (BT-474, MDA-MB-453). Transgene expression was detected in brain homogenates of Rag1 knockout mice following a single ICV injection of AAV9.UbC.trastuzumab (1 × 1011 vector genome copies [GC]/mouse) and tumor progression was inhibited in xenograft models of breast-to-brain metastasis. In NHPs, ICM delivery of AAV9.UbC.trastuzumab (3 × 1013 GC/animal) was well tolerated (36-37 days in-life) and resulted in transgene expression in CNS tissues and cerebrospinal fluid at levels sufficient to induce complete tumor remission in MDA-MB-453 brain xenografts. With AAV9's proven clinical safety record, this gene therapy may represent a viable approach for targeting HER2 + CNS malignancies.

Drosophila Importin Alpha 1 (Dα1) Is Required to Maintain Germline Stem Cells in the Testis Niche.

Stem cell maintenance and differentiation can be regulated via the differential activity of transcription factors within stem cells and their progeny. For these factors to be active, they need to be transported from their site of synthesis in the cytoplasm into the nucleus. A tissue-specific requirement for factors involved in nuclear importation is a potential mechanism to regulate stem cell differentiation. We have undertaken a characterization of male sterile importin alpha 1 (Dα1) null alleles in Drosophila and found that Dα1 is required for maintaining germline stem cells (GSCs) in the testis niche. The loss of GSCs can be rescued by ectopic expression of Dα1 within the germline but the animals are still infertile, indicating a second role for Dα1 in spermatogenesis. Expression of a Dα1 dominant negative transgene in GSCs confirmed a functional requirement for Dα1 in GSC maintenance but expression of the transgene in differentiating spermatogonia did not exhibit a phenotype indicating a specific role for Dα1 within GSCs. Our data indicate that Dα1 is utilized as a regulatory protein within GSCs to facilitate nuclear importation of proteins that maintain the stem cell pool.

Vector integration and fate in the hemophilia dog liver multiple years after AAV-FVIII gene transfer

AbstractGene therapy using adeno-associated virus (AAV) vectors is a promising approach for the treatment of monogenic disorders. Long-term multiyear transgene expression has been demonstrated in animal models and clinical studies. Nevertheless, uncertainties remain concerning the nature of AAV vector persistence and whether there is a potential for genotoxicity. Here, we describe the mechanisms of AAV vector persistence in the liver of a severe hemophilia A dog model (male = 4, hemizygous; and female = 4, homozygous), more than a decade after portal vein delivery. The predominant vector form was nonintegrated episomal structures with levels correlating with long-term transgene expression. Random integration was seen in all samples (median frequency, 9.3e−4 sites per cell), with small numbers of nonrandom common integration sites associated with open chromatin. No full-length integrated vectors were found, supporting predominant episomal vector-mediated long-term transgene expression. Despite integration, this was not associated with oncogene upregulation or histopathological evidence of tumorigenesis. These findings support the long-term safety of this therapeutic modality.

Protein phosphatase 2A anchoring disruptor gene therapy for familial dilated cardiomyopathy

Familial Dilated Cardiomyopathy is a prevalent cause of heart failure that results from the mutation of genes encoding proteins of diverse function. Despite modern therapy, Dilated Cardiomyopathy typically has a poor outcome and is the leading cause of cardiac transplantation. The phosphatase PP2A at cardiomyocyte perinuclear mAKAPβ signalosomes promotes pathological eccentric cardiac remodeling, as is characteristic of Dilated Cardiomyopathy. Displacement of PP2A from mAKAPβ, inhibiting PP2A function in that intracellular compartment, can be achieved by expression of a mAKAPβ-derived PP2A binding domain-derived peptide. To test whether PP2A anchoring disruption would be effective at preventing Dilated Cardiomyopathy-associated cardiac dysfunction, the adeno-associated virus gene therapy vector AAV9sc.PBD was devised to express the disrupting peptide in cardiomyocytes in vivo. Proof-of-concept is now provided that AAV9sc.PBD improves the cardiac structure and function of a cardiomyopathy mouse model involving transgenic expression of a mutant α-tropomyosin E54K Tpm1 allele, while AAV9sc.PBD has no effect on normal non-transgenic mice. At the cellular level, AAV9sc.PBD restores cardiomyocyte morphology and gene expression in the mutant Tpm1 mouse. As the mechanism of AAV9sc.PBD action suggests potential efficacy in Dilated Cardiomyopathy regardless of the underlying etiology, these data support the further testing of AAV9sc.PBD as a broad-based treatment for Dilated Cardiomyopathy.

Trisomy silencing by XIST: translational prospects and challenges.

XIST RNA is heavily studied for its role in fundamental epigenetics and X-chromosome inactivation; however, the translational potential of this singular RNA has been much less explored. This article combines elements of a review on XIST biology with our perspective on the translational prospects and challenges of XIST transgenics. We first briefly review aspects of XIST RNA basic biology that are key to its translational relevance, and then discuss recent efforts to develop translational utility of XIST for chromosome dosage disorders, particularly Down syndrome (DS). Remarkably, it was shownin vitrothat expression of an XIST transgene inserted into one chromosome 21 can comprehensively silence that chromosome and "dosage compensate" Trisomy 21, the cause of DS. Here we summarize recent findings and discuss potential paths whereby ability to induce "trisomy silencing" can advance translational research for new therapeutic strategies. Despite its common nature, the underlying biology for various aspects of DS, including cell types and pathways impacted (and when), is poorly understood. Recent studies show that an inducible iPSC system to dosage-correct chromosome 21 can provide a powerful approach to unravel the cells and pathwaysdirectlyimpacted, and the developmental timing, information key to design pharmacotherapeutics. In addition, we discuss prospects of a more far-reaching and challenging possibility that XIST itself could be developed into a therapeutic agent, for targeted cellular "chromosome therapy". A few rare case studies of imbalanced X;autosome translocations indicate that natural XIST can rescue an otherwise lethal trisomy. The potential efficacy of XIST transgenes later in development faces substantial biological and technical challenges, although recent findings are encouraging, and technology is rapidly evolving. Hence, it is compelling to consider the transformative possibility that XIST-mediated chromosome therapy may ultimately be developed, for specific pathologies seen in DS, or other duplication disorders.

Next-generation strategies to improve safety and efficacy of adeno-associated virus-based gene therapy for hemophilia: lessons from clinical trials in other gene therapies.

Three major directions for the global progress of adeno-associated virus (AAV) vectors for gene therapies (GT) are analyzed: a) engineering vectors to increase transgene expression; b) aligning interests of the health system with costs and challenges for pharmaceutical industry; c) refining patient eligibility criteria, and endpoints definition. Currently employed AAV vectors may cause toxicity and adverse events. Furthermore, studies in animals do not fully predict risks and clinical benefits of AAV-based GT, and animal models reflecting the heterogeneity of certain clinical settings (e.g., congestive heart failure) are poorly available for improving AAV-based GT. Finally, antisense and gene editing approaches will soon complement gene augmentation strategies for the stable solution of unsolved issues of AAV-based GT. While minimizing toxicity, next-generation AAV vectors should decrease the viral load needed to achieve therapeutic efficacy; be functional in a restricted cellular subset; avoid transgene expression in unwanted cells (e.g., hepatocytes), and escape immune oversight in AAV-based GT. The role of stress-induced apoptosis in the loss of transgene expression in GT should be also explored. Aligning interests and obligations of pharmaceutical industry with those of the health system is critical for AAV-based GT success. Costs and challenges for pharmaceutical industry include a) removing impurities from AAV; b) validating tests to measure treatment efficacy, c) promoting training programs to standardize vector genomes delivery, d) collecting long-term follow-up data, and e) maintaining sustainability and cost-effectiveness of AAV-based GT. In rare disorders with small patient numbers (e.g., hemophilia), clearcut outcomes are mandatory as endpoints of unequivocal efficacy data.