Low Transduction Efficiency Research Articles

Ultra-sensitive immobilization-free homogeneous electrochemiluminescence biosensor for thrombin detection via electrostatic interaction and Exo I-powered signal amplification

Herein, we present the development of an ultra-sensitive immobilization-free homogeneous electrochemiluminescence (ECL) biosensor, leveraging the electrostatic repulsion between a negatively charged indium tin oxide (ITO) electrode and DNA and exonuclease I (Exo I)-powered signal amplification, to achieve highly efficient detection of thrombin. Specifically, the aptamer and the complementary DNA engage in the formation of a double-stranded DNA (dsDNA) complex. This negatively charged dsDNA structure subsequently associates with a positively charged ECL indicator, namely ruthenium phenanthroline (Ru(phen)32+), resulting in the generation of the dsDNA-Ru(phen)32+ ECL probe. The negatively charged dsDNA-Ru(phen)32+ experiences electrostatic repulsion from the negatively charged ITO electrode, resulting in a low ECL signal. Nonetheless, upon the addition of thrombin, the aptamer preferentially binds to thrombin, triggering the releases of the embedded Ru(phen)32+ facilitated by Exo I and hence resulting in a robust and enhanced ECL signal. The amplified ECL signal is linearly correlated with the logarithm of thrombin concentration within a detection range spanning from 10 fmol/mL to 50 pmol/mL, with a remarkable detection limit of 3.21 fmol/mL. This strategy eliminates the need for cumbersome labeling steps, avoids the electrode modification process, overcoming the low immobilization efficiency of aptamers and poor signal transduction of indicators labeled at the end of DNA.

CRISPR/Cas-mediated “one to more” lighting-up nucleic acid detection using aggregation-induced emission luminogens

CRISPR diagnostics are effective but suffer from low signal transduction efficiency, limited sensitivity, and poor stability due to their reliance on the trans-cleavage of single-stranded nucleic acid fluorescent reporters. Here, we present CrisprAIE, which integrates CRISPR/Cas reactions with “one to more” aggregation-induced emission luminogen (AIEgen) lighting-up fluorescence generated by the trans-cleavage of Cas proteins to AIEgen-incorporated double-stranded DNA labeled with single-stranded nucleic acid linkers and Black Hole Quencher groups at both ends (Q-dsDNA/AIEgens-Q). CrisprAIE demonstrates superior performance in the clinical nucleic acid detection of norovirus and SARS-CoV-2 regardless of amplification. Moreover, the diagnostic potential of CrisprAIE is further enhanced by integrating it with spherical nucleic acid-modified AIEgens (SNA/AIEgens) and a portable cellphone-based readout device. The improved CrisprAIE system, utilizing Q-dsDNA/AIEgen-Q and SNA/AIEgen reporters, exhibits approximately 80- and 270-fold improvements in sensitivity, respectively, compared to conventional CRISPR-based diagnostics. We believe CrisprAIE can be readily extended as a universal signal generation strategy to significantly enhance the detection efficiency of almost all existing CRISPR-based diagnostics.

AAV-mediated gene delivery to the cardiac conduction system

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): NWO open technology program Biopace #18485 Background Gene therapy for cardiac arrhythmias has so far mostly focused on modifying the arrhythmogenic substrate in working myocardium or on creating biological pacemakers with viral vectors that did not selectively target the cardiac conduction system (CCS). As a result, CCS targeting by viral vectors is a relatively unexplored area. Aim In the current project, we explored the basic requirements for CCS targeting using adeno-associated virus (AAV) vectors. Methods Regular AAV9 and AAV-Myo4A capsid-pseudotyped AAV2 vectors (hereafter called AAV2/9 and AAV2/Myo4A vectors, respectively) expressing enhanced green fluorescent protein (eGFP) from a CMV or TNNT2 promoter, respectively, and an AAV2/9 vector expressing eGFP from a human GJA5 regulatory element were systemically injected in mice. After 4 weeks, hearts were harvested, fixed with paraformaldehyde and stained for TNNT2, eGFP, HCN4 and DNA to determine CCS transduction. Results Transduction of the sinoatrial and atrioventricular (AV) node using AAV2/9 vectors was very poor, while transduction was visible in the atrioventricular (i.e. His) bundle and working myocardium. In contrast, the transduction rate of the AV node and bundle using AAV2/Myo4A vectors were similar to that of the working myocardium. The human GJA5 regulatory element was able to restrict eGFP expression by AAV2/9 vectors to the AV bundle in 2/3 animals but the transduction efficiency was low. A third animal showed no transduction of any tissue, either indicative of the low transduction efficiency or of a failed injection. Conclusion This study not only provides proof-of-principle for CCS-targeted gene therapy but also shows that CCS transduction can be improved through AAV capsid engineering and be restricted to (parts of) the CCS by using specific transcriptional control elements.

Targeted gene delivery systems for T-cell engineering.

T lymphocytes are indispensable for the host systems of defense against pathogens, tumors, and environmental threats. The therapeutic potential of harnessing the cytotoxic properties of T lymphocytes for antigen-specific cell elimination is both evident and efficacious. Genetically engineered T-cells, such as those employed in CAR-T and TCR-T cell therapies, have demonstrated significant clinical benefits in treating cancer and autoimmune disorders. However, the current landscape of T-cell genetic engineering is dominated by strategies that necessitate in vitro T-cell isolation and modification, which introduce complexity and prolong the development timeline of T-cell based immunotherapies. This review explores the complexities of gene delivery systems designed for T cells, covering both viral and nonviral vectors. Viral vectors are known for their high transduction efficiency, yet they face significant limitations, such as potential immunogenicity and the complexities involved in large-scale production. Nonviral vectors, conversely, offer a safer profile and the potential for scalable manufacturing, yet they often struggle with lower transduction efficiency. The pursuit of gene delivery systems that can achieve targeted gene transfer to T cell without the need for isolation represents a significant advancement in the field. This review assesses the design principles and current research progress of such systems, highlighting the potential for in vivo gene modification therapies that could revolutionize T-cell based treatments. By providing a comprehensive analysis of these systems, we aim to contribute valuable insights into the future development of T-cell immunotherapy.

Glycosylation of recombinant adeno-associated virus serotype 6

Glycosylation of biopharmaceuticals can affect their safety and efficacy. Glycans can occur on recombinant adeno-associated viruses (rAAVs) that are used for gene therapy; however, the types of glycan that attach to rAAVs are controversial. Here, we conducted lectin microarray analyses on six rAAVs serotype 6 (rAAV6) preparations that were produced differently. We demonstrate that O-glycans considered to be attached to rAAV6 were recognized by Agaricus bisporus agglutinin (ABA) and that N-glycans were detected in rAAV6 purified without affinity-chromatography. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that the N-glycans detected in rAAV6 were derived from host cell proteins. A combination of ABA-based fractionation and LC-MS/MS revealed that rAAV6 was O-glycosylated with the mucin-type glycans, O-GalNAc (Tn-antigen) and mono- and di-sialylated Galβ1-3GalNAc (T-antigen) at S156, T162, T194, and T201 in viral protein (VP) 2, and with O-GlcNAc at T242 in VP3. The mucin-type O-glycosylated rAAV6 particles were 0.1%–1% of total particles. Further physicochemical and biological analyses revealed that mucin-type O-glycosylated rAAV6 had a lower ratio of VP1 to VP2/VP3, resulting in lower transduction efficiency both in vitro and in vivo compared with rAAV6 without mucin-type O-glycans. This report details conclusive evidence of rAAV glycosylation and its impact on rAAV-based therapeutics.

Gene Therapy for Cardiovascular Disease: Clinical Perspectives.

Cardiovascular disease (CVD) stands as one of the leading causes of death in the United States, with its prevalence steadily on the rise. Traditional therapeutic approaches, such as pharmacological treatment, cardiovascular intervention, and surgery, have inherent limitations. In response to these challenges, cardiac gene therapy has emerged as a promising alternative for treating CVD patients. However, several obstacles persist, including the low efficiency of gene transduction, immune reactions to vectors or transduced cells, and the occurrence of off-target effects. While preclinical research has demonstrated significant success in various CVD model in both small and large animals, the translation of these findings to clinical applications has, for the most part, yielded disappointing results, except for some early, albeit small, trials. This review aims to provide a comprehensive summary of recent preclinical and clinical studies on gene therapy for various CVDs. Additionally, we discuss the existing limitations and challenges that hinder the widespread clinical application of cardiac gene therapy.

Bleomycin promotes rAAV2 transduction via DNA-PKcs/Artemis-mediated DNA break repair pathways

Because it is safe and has a simple genome, recombinant adeno-associated virus (rAAV) is an extremely appealing vector for delivery in in vivo gene therapy. However, its low transduction efficiency for some cells, limits its further application in the field of gene therapy. Bleomycin is a chemotherapeutic agent approved by the FDA whose effect on rAAV transduction has not been studied. In this study, we systematically investigated the effect of Bleomycin on the second-strand synthesis and used CRISPR/CAS9 and RNAi methods to understand the effects of Bleomycin on rAAV vector transduction, particularly the effect of DNA repair enzymes. The results showed that Bleomycin could promote rAAV2 transduction both in vivo and in vitro. Increased transduction was discovered to be a direct result of decreased cytoplasmic rAAV particle degradation and increased second-strand synthesis. TDP1, PNKP, and SETMAR are required to repair the DNA damage gap caused by Bleomycin, TDP1, PNKP, and SETMAR promote rAAV second-strand synthesis. Bleomycin induced DNA-PKcs phosphorylation and phosphorylated DNA-PKcs and Artemis promoted second-strand synthesis. The current study identifies an effective method for increasing the capability and scope of in-vivo and in-vitro rAAV applications, which can amplify cell transduction at Bleomycin concentrations. It also supplies information on combining tumor gene therapy with chemotherapy.

B Cell Therapeutics

Plasma cells are specialized immune cells that can survive in the bone marrow for decades and continually secrete large amounts of antibodies, thus providing long-term, often lifelong protection from repeated exposures to a pathogen. The long-term survival and the capability to secrete high amounts of protein make plasma cells a potential candidate for stable delivery of therapeutic proteins and antibodies. However, engineering plasma cells or B cells has been challenging due to low lentiviral transduction efficiency, incompatibility with plasmid transfection and loss of episomal DNA expression over time. We have previously shown that human plasma cells can be engineered to secrete antibodies and proteins in in vitro culture systems via gene editing. In the current study, we have generated engineered murine plasma cells that can secrete the clotting factor IX (FIX). We hypothesize that the FIX-secreting plasma cells will engraft long-term in FIX-deficient immunocompetent mice and secrete therapeutic levels of FIX leading to protection from Hemophilia B. The standard of care for Hemophilia B is repeated intravenous infusion of recombinant FIX or AAV-based gene therapy. However, 30-60% humans have pre-existing immunity to AAV-based gene therapy and gene therapy can induce an immune response in people lacking pre-existing antibodies. Moreover, gene therapy is unsuitable for pediatric patients due to their rapid growth and proportional loss of AAV. Plasma cell-based therapy is lowly immunogenic and can be used for redosing in pediatric patients that require more product with age. Here, we will describe proof-of-concept studies showing that engineered murine plasma cells can engraft into immunocompetent mice without the need for conditioning and produce stable levels of protein for >10 weeks. We developed a three-step in vitro co-culture system that can be used to engineer and differentiate naïve B cells into plasma cells. These in vitro generated murine plasma cells had a comparable gene expression profile and phenotype to natural plasma cells and can engraft stably, in the absence of conditioning, into immunocompetent mice whereupon they secreted antibodies (Figure 1). Moreover, we show that plasma cells can be re-dosed to increase antibody levels without triggering a secondary immune response (Figure 1). For delivery of therapeutic proteins, we have developed adeno-associated virus-based homology directed repair reagents to edit murine B cells at several loci, including beta-2 microglobulin, Rosa-26, and the antibody heavy chain. By co-delivery of cis-linked fluorescent markers, we were able to sort and expand only edited cells, enabling enrichment of cells expressing a therapeutic protein. In proof-of-concept studies, we have used this approach to generate a human FIX-secreting plasma cell product with close to 90% edited cells at the end of the B cell culture. The enriched plasma cells generated 0.28 pg FIX/cell/day compared to 0.19 pg FIX/cell/day generated by the bulk edited cells (figure 2). We estimate that engraftment of 1 million edited plasma cells is sufficient to generate therapeutic levels of FIX upon (figure 2). We have developed reagents for a novel cell-based therapeutic tool with protein secretion capabilities and far-reaching applications. Out studies will provide insights into the feasibility and efficacy of a B cell based therapeutic product.

Type I Interferon Blockade Enhances Transduction Efficiency and Efficacy of Non-Activated CAR T Cells

Chimeric antigen receptor (CAR) T-cell therapies have revolutionized the treatment of advanced hematologic cancers, showing impressive response rates in pivotal trials. Despite this success, challenges in CAR T-cell adoption persist due to tumor resistance mechanisms, manufacturing failures, and limitations in CAR T-cell persistence and effector function. Strategies that promote T-cell persistence, proliferation, and cytotoxic activity are essential to improving long-term outcomes of CAR T therapy. The most common protocol for CAR T-cell manufacturing involves lentiviral vector transduction of activated T-cells followed by ex vivo culture. However, this approach may drive terminal T-cell differentiation and exhaustion, compromising therapeutic efficacy. In contrast, non-activated CAR T-cell manufacturing has shown promise in enhancing T-cell differentiation and tumor control. Yet, the low transduction efficiency of quiescent T-cells remains a challenge. In this study, we explore the role of type I interferon (IFN)-mediated innate immune responses in limiting CAR T manufacturing efficiency. We hypothesize that blocking these responses using the vaccinia virus-encoded type I interferon-binding protein can enhance transduction efficiency and promote less-differentiated CAR T-cell phenotypes, including naïve and central memory-like differentiation. Our findings demonstrate that lentiviral vector transduction triggers an innate immune response in non-activated T-cells, resulting in reduced transduction efficiency, and altered T-cell differentiation. Inhibiting the anti-viral response via anti-IFN leads to improved T-cell differentiation and exhaustion characteristics. Importantly, anti-IFN exposure supports the enrichment of naive-like T cells: a subset noted for superior clinical efficacy. Anti-IFN also reduces LAG3 expression. Collectively, these findings position anti-IFN as a novel means to enhance CAR T-cell persistence and anti-tumor function. We further investigate the influence of sustained anti-IFN exposure by designing lentiviral transfer plasmids encoding both a CAR and secreted type I interferon-binding protein. These cells showed enhanced cytolytic activity and cytokine production against target cells. Our studies in mice show promising results, suggesting that the protein secretion enhance therapeutic activity against tumors employing sustained type I interferon signaling for immune escape. Overall, targeting the type I interferon response during non-activated CAR T-cell manufacturing offers a potential solution to improve transduction efficiency and enhance CAR T-cell functionality. Given the therapeutic promise of non-activated CAR T cells, our findings have immediate translational relevance for broadening their appeal against cancer. Figure 1. Exposure to anti-IFN increases transduction efficiency and naïve-like differentiation of lentivirus-transduced non-activated T-cells A) Freshly isolated human T-cells were incubated in media with aIFN or vehicle, for one hour at room temperature. The cells were resuspended in complete media and then transduced with lentiviral vector encoding iRFP. Expression of iRFP was evaluated 96 h after transduction by flow cytometry. b, Human T-cells were isolated and prepared as above. At 96 h post-transduction, cells were stained with anti-CD45RO and anti-CCR7 antibodies and samples were evaluated by flow cytometry. Data for A and B are representative of multiple experiments with three independent donors.

Adaptive energy harvesting from low-level ambient vibrations

Harvesting energy from ambient mechanical vibrations is an enabling technology with profound societal impacts. However, existing methods suffer from two major technical deficiencies: the weak coupling between the harvester and the vibrating source and the low energy transduction efficiency. Here we show an adaptive approach can overcome these challenges, leading to improvements by orders of magnitude. Using a piezoelectric device, we demonstrate that a conservative actuation force can block energy from flowing back to the vibrating source, thereby increasing the coupling strength and increasing the transduction efficiency significantly. Using low actuation forces, the adaptive approach can extract almost two orders of magnitude more power than the traditional approach from a multi-frequency vibration. The improvement can increase to three orders of magnitude when moderate actuation force is used. If materials that can withstand a high actuation voltage are available, the adaptive approach has the potential to surpass the theoretical performance ceiling of the traditional approach, improving the upper-bound performance by over 100 times and creating a significant opportunity for the technology to advance.

Enhanced sensitivity of neutralizing antibody detection for different AAV serotypes using HeLa cells with overexpressed AAVR.

A cell-based transduction inhibition assay (TI) is widely used in clinical trials to detect neutralizing antibody (NAb) titers against recombinant adeno-associated virus (rAAV), one of the most important criteria to exclude patients in gene therapy. Different cell lines are used in cell-based TI because the rAAV transduction efficiencies vary largely among serotypes. A cell line suitable for TI for most serotypes is highly desirable, especially for those with very low transduction efficiencies in vitro such as rAAV8 and rAAV9. Herein, we report an AAVR-HeLa, a stable cell line with overexpressed AAVR, a newly identified receptor for rAAVs, was established for cell-based TIs. The AAVR expression level in AAVR-HeLa cells was approximately 10-fold higher than in HeLa cells, and was stably transfected after twenty three passages. For all AAV serotypes (AAV1-10), except for AAV4, the transduction efficiencies increased significantly in AAVR-HeLa cells. It was demonstrated that the AAVR enhancement of transduction efficiency was only for rAAV and not for lentiviral and adenoviral vectors. According to the minimal multiplicity of infection (MOIs) for the assay, the NAb detection sensitivity increased at least 10 and 20 fold for AAV8 and AAV9, respectively. The seroprevalence of NAbs were investigated at the 1:30 level as a cutoff value using AAVR-HeLa cells. It was shown that the seropositive rate for AAV2 was 87% in serum samples from 99 adults, followed by lower seropositive rates for AAV5 (7%), AAV8 (7%) and AAV9 (1%). Venn diagram analysis showed the presence of cross-reactivity of NAbs to two or three serotypes in 13 samples (13.1%). However, no patient was found to possess NAbs for all the four serotypes. These results demonstrated that the AAVR-HeLa cell line may be utilized to detect the NAbs through cell-based TI assays for most of AAV serotypes.

Adeno-Associated virus 8 delivers an immunomodulatory peptide to mouse liver more efficiently than to rat liver.

Targeting the Kv1.3 potassium channel has proven effective in reducing obesity and the severity of animal models of autoimmune disease. Stichodactyla toxin (ShK), isolated from the sea anemone Stichodactyla helianthus, is a potent blocker of Kv1.3. Several of its analogs are some of the most potent and selective blockers of this channel. However, like most biologics, ShK and its analogs require injections for their delivery, and repeated injections reduce patient compliance during the treatment of chronic diseases. We hypothesized that inducing the expression of an ShK analog by hepatocytes would remove the requirement for frequent injections and lead to a sustained level of Kv1.3 blocker in the circulation. To this goal, we tested the ability of Adeno-Associated Virus (AAV)8 vectors to target hepatocytes for expressing the ShK analog, ShK-235 (AAV-ShK-235) in rodents. We designed AAV8 vectors expressing the target transgene, ShK-235, or Enhanced Green fluorescent protein (EGFP). Transduction of mouse livers led to the production of sufficient levels of functional ShK-235 in the serum from AAV-ShK-235 single-injected mice to block Kv1.3 channels. However, AAV-ShK-235 therapy was not effective in reducing high-fat diet-induced obesity in mice. In addition, injection of even high doses of AAV8-ShK-235 to rats resulted in a very low liver transduction efficiency and failed to reduce inflammation in a well-established rat model of delayed-type hypersensitivity. In conclusion, the AAV8-based delivery of ShK-235 was highly effective in inducing the secretion of functional Kv1.3-blocking peptide in mouse, but not rat, hepatocytes yet did not reduce obesity in mice fed a high-fat diet.

Characterization of a Bioengineered AAV3B Capsid Variant with Enhanced Hepatocyte Tropism and Immune Evasion.

Capsid engineering of adeno-associated virus (AAV) can surmount current limitations to gene therapy such as broad tissue tropism, low transduction efficiency, or pre-existing neutralizing antibodies (NAb) that restrict patient eligibility. We previously generated an AAV3B combinatorial capsid library by integrating rational design and directed evolution with the aim of improving hepatotropism. A potential isolate, AAV3B-DE5, gained a selective proliferative advantage over five rounds of iterative selection in hepatocyte spheroid cultures. In this study, we reanalyzed our original dataset derived from the AAV3B combinatorial library and isolated variants from earlier (one to three) rounds of selection, with the assumption that variants with faster replication kinetics are not necessarily the most efficient transducers. We identified a potential candidate, AAV3B-V04, which demonstrated significantly enhanced transduction in mouse-passaged primary human hepatocytes as well as in humanized liver chimeric mice, compared to the parental AAV3B or the previously described isolate, AAV3B-DE5. Interestingly, the AAV3B-V04 capsid variant exhibited significantly reduced seroreactivity to pooled or individual human serum samples. Forty-four percent of serum samples with pre-existing NAbs to AAV3B had 5- to 20-fold lower reciprocal NAb titers to AAV3B-V04. AAV3B-V04 has only nine amino acid substitutions, clustered in variable region IV compared to AAV3B, indicating the importance of the loops at the top of the three-fold protrusions in determining both transduction efficiency and immunogenicity. This study highlights the effectiveness of rational design combined with targeted selection for enhanced AAV transduction via molecular evolution approaches. Our findings support the concept of limiting selection rounds to isolate the best transducing AAV3B variant without outgrowth of faster replicating candidates. We conclude that AAV3B-V04 provides advantages such as improved human hepatocyte tropism and immune evasion and propose its utility as a superior candidate for liver gene therapy.

Limitations and Overcomes of CAR-NK for Cancer Immunotherapy

Adoptive cell treatment utilising chimeric antigen receptor (CAR) immunotherapy is gaining remarkable breakthroughs. In solid tumours, CAR immunotherapy lags far behind. CAR T cell production, does not have tumor-specific antigens, and is not effective in CAR T cell infiltration and trafficking into tumour sites, therapy-associated toxicity, immunosuppressive TME, and antigen escape are among the primary obstacles for CAR immunotherapy in solid tumours. Compared to CAR T cells, CAR NK cells have a number of benefits, including the ability to be processed from preexisting cell lines with mismatched MHC, the ability to kill bad cells via CAR-independent and CAR-dependent pathway with less toxicity. One clinical study demonstrated the effectiveness and safety of CAR NK cell treatment. However, CAR NK cell therapy also has its drawbacks, including, low persistence, lack of transporting pathway, immunosuppressive tumour microenvironment, and low lentivirus transduction efficiency. Solving these issues are significantly important for this technology to be accepted in future use.

The use of melittin to enhance transgene expression mediated by recombinant adeno-associated virus serotype 2 vectors both in vitro and in vivo

ObjectiveMelittin, a cell-penetrating peptide, improves the efficiency of many non-viral gene delivery vectors, yet its application in viral vectors has not been well studied. The non-pathogenic recombinant adeno-associated virus (rAAV) vector is an ideal in vivo gene delivery vector. However, its full potential will only be achieved after improvement of its transduction efficiency. To improve the transduction efficiency of rAAV2 vectors, we attempted to develop a melittin-based rAAV2 vector delivery strategy. MethodsThe melittin peptide was inserted into the rAAV2 capsid either in the loop VIII of all viral proteins (VPs) or at the N terminus of VP2. Various rAAV2-gfp or -fluc vectors were subjected to quantitative real-time polymerase chain reaction and Western blot assays to determine their titers and integrity of capsid proteins, respectively. Alternatively, the vectors based on wild-type capsid were pre-incubated with melittin, followed by transduction of cultured cells or tail vein administration of the mixture to C57BL/6 and BALB/c nude mice. In vivo bioluminescence imaging was performed to evaluate the transgene expression. ResultsrAAV2 vectors with melittin peptide inserted in the loop VIII of VPs had low transduction efficiency, probably due to dramatically reduced ability to bind to the target cells. Fusing the melittin peptide at the N-terminus of VP2 produced vectors without the VP2 subunit. Interestingly, among the commonly used rAAV vectors, pre-incubation of rAAV2 and rAAV6 vectors with melittin significantly enhanced their transduction efficiency in HEK293 and Huh7 cells in vitro. Melittin also had the ability to increase the rAAV2-mediated transgene expression in mouse liver in vivo. Mechanistically, melittin did not change the vector-receptor interaction. Moreover, cell counting kit-8 assays of cultured cells and serum transaminase levels indicated melittin had little cytotoxicity. ConclusionPre-incubation with melittin, but not insertion of melittin into the rAAV2 capsid, significantly enhanced rAAV2-mediated transgene expression. Although further in vivo evaluations are required, this research not only expands the pharmacological potential of melittin, but also provides a new strategy to improve gene therapy mediated by rAAV vectors.

CRISPR/Cas9 ribonucleoprotein (RNP) complex enables higher viability of transfected cells in genome editing of acute myeloid cells.

BackgroundClustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has become an increasingly vital tool for modifying gene expression in a variety of cell types. Lentiviral transduction and electroporation are the two main approaches used to deliver CRISPR/Cas9 into cells. However, the application of CRISPR/Cas9 in primary hematopoietic cells has been limited due to either low transduction efficiency in terms of viral-based delivery or difficult selection and enrichment of transfected and edited cells with respect to electroporation of CRISPR/Cas9 ribonucleoprotein (RNP).MethodsIn this study in vitro transcription was used to synthesize the guide RNA (gRNA), and plasmid pL-CRISPR.EFS.GFP was used as its DNA template. Then the in vitro transcribed gRNA was labeled with pCp-Cy5 via T4 ligase before incubating with Cas9 protein. Furthermore, CRISPR/Cas9 RNP was electroporated into primary CD34+ cells isolated from cord blood, and cell survival rate and transfection efficiency were calculated and compared to that of lentiviral transduction.ResultsHere, we show that electroporation of CRISPR/Cas9 RNP resulted in higher cell viability compared to electroporation of CRISPR/Cas9 all-in-one plasmid, providing important findings for further studies in hematology via CRISPR/Cas9 technology. Moreover, we established a method for labeling in vitro-transcribed gRNA with fluorophore and the sorted fluorescent cells displayed higher knockout efficiency than nonsorted transfected cells.ConclusionsElectroporation of fluorescence labeled CRISPR/Cas9 RNP is a perspective approach of gene editing. Our study provides an efficient and time-saving approach for genome-editing in hematopoietic cells.

Organoid transduction using recombinant adeno‐associated viral vectors: Challenges and opportunities

Cellular 3D structures, for example, organoids, are an excellent model for studying and developing treatments for various diseases, including hereditary ones. Therefore, they are increasingly being used in biomedical research. From the point of view of safety and efficacy, recombinant adeno-associated viral (rAAV) vectors are currently most in demand for the delivery of various transgenes for gene replacement therapy or other applications. The delivery of transgenes using rAAV vectors to various types of organoids is an urgent task, however, it is associated with a number of problems that are discussed in this review. Cellular heterogeneity and specifics of cultivation of 3D structures determine the complexity of rAAV delivery and are sometimes associated with low transduction efficiency. This review surveys the main ways to solve emerging problems and increase the efficiency of transgene delivery using rAAVs to organoids. A clear understanding of the stage of development of the organoid, its cellular composition and the presence of surface receptors will allow obtaining high levels of organoid transduction with existing rAAV vectors.

Abstract CT522: Feasibility and safety of a novel CD19 CAR T cell therapy in adults with R/R B-NHL

Abstract Genetically engineered chimeric antigen receptor (CAR) T cells have exhibited distinct effectiveness against chemotherapy refractory CD19 expressing B cell malignancies in both adults and children. This phase I clinical trial tests a novel anti-CD19 CAR T cell product in adults with relapsed/refractory (R/R) B-cell Non-Hodgkin’s Lymphoma (B-NHL). The CAR construct is comprised of the short chain variable regions of the anti-CD19 monoclonal antibody FMC63, the TNFRSF19-derived transmembrane domain, the 4-1BB costimulatory domain, and the CD3-zeta signaling domain. CD19 CAR T cells were manufactured utilizing the CliniMACS Prodigy® T Cell Transduction Process (CD3/CD28 TransAct™ reagent) allowing for highly automated production, with IL-7 and IL-15 used for T cell expansion for 8-12 days. To date, 7 patients have been treated with an average dose of 1.2 ± 0.2 x 108 CAR T cells. The histology includes marginal zone lymphoma (n=1), follicular lymphoma Grade IIIA (n=1), transformed lymphoma (n=3), follicular lymphoma low grade (n=2), and diffuse large B-cell lymphoma (n=1). One patient required a second apheresis due to poor cell expansion. Despite heterogeneity in disease subtype and leukapheresis product quality, CAR T production and expansion have been consistent with final transduction efficiencies between 14-45%, cell viability between 88-91%, and an overall average yield of 3.2 ± 0.3 x 109 cells before harvest, allowing for product banking. No safety-related out of specifications (OOS) events have occurred, however, 2 patients had OOS product infused due to low transduction efficiency (both at 14% rather than the ≥ 20% release criteria). Two patients experienced Grade 2 CRS, 1 patient experienced Grade 2 neurotoxicity; otherwise, no new safety signals were detected. Disease response was assessed on Days 90, 180, 270, and 360 post-infusion. The assessments were based on 2014 Lugano criteria. Even with 2 OOS products, Day 90 scans showed a complete metabolic remission (CMR) in 6 evaluable patients to date. Of the 6 patients with CMR, 1 patient progressed at Day 180 and the others remain in remission (median f/u = 12 months). Flow cytometry was utilized to measure CAR T cell peak expansion and persistence in 5 patients. Peak CAR T cell expansion (2.9-44.4% of CD3 cells) ranged from Day 5 to 15. Cell persistence was detected for the 5 patients through at least Day 180. ddPCR is currently in development to perform persistence testing in parallel. Additionally, cytokine concentrations including INFγ, IL-10, IL-12p70, IL-13, IL1β, IL-2, IL-4, IL-6, IL-8, and TNFα were evaluated over the first 30 days. Overall, 7 patients diagnosed with 5 different B-NHL subtypes have been treated with the CD19 CAR T cell product. Manufacturing was successful for all patients with no safety related OOS, and no new post-infusion safety signals detected. To date, 6 out of 7 patients are alive, 5 with CMR and with CD19 CAR T cell persistence through at least 180 days. Citation Format: Manali Kamdar, Cheri Adams, Steven Bair, Boro Dropulic, Jonathon Gutman, Bradley Haverkos, Kimberly Jordan, Rebecca Mallo, Russell Marians, Felicia Mast, Lindsey Murphy, Andrew Roth, Matthew Seefeldt, Andrew Worden, Mike Kadan, Ying Xiong, Dina Schneider, Rimas Orentas, Terry Fry, Michael Verneris. Feasibility and safety of a novel CD19 CAR T cell therapy in adults with R/R B-NHL [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 CT522.

Optimizing rAAV6 transduction of primary T cells for the generation of anti-CD19 AAV-CAR-T cells.

Recombinant Adeno-associated virus(rAAV) is currently the most widely used gene delivery vector and has been successfully used in various disease models, benefiting from its low immunogenicity, almost no toxicity, and no reported pathogenicity in humans. However, its low transduction efficiency for primary cells, especially for T lymphocytes, limits its further application in the field of cell therapy. In this study, we optimized the protocol for rAAV6 transduction of primary T cells, significantly improved the expression efficiency of the rAAV6 delivered CAR gene, and successfully generated rAAV6-based CAR-T cells (AAV-CAR-T). The gene expression intensity (mean fluorescence intensity, MFI) of rAAV6 transduced T cells treated with the tyrosine kinase inhibitor, Genistein, was increased 1-3-fold. Moreover, our results showed that rAAV6 efficiently transduced T cells stimulated with OKT3 and the gene expression could be enhanced 3-fold with an OKT3 concentration of 50ng/mL in the medium. The gene expression intensity of T cells treated with OKT3 together with genistein could be augmented by 7-fold. Based on the above-optimized method, CAR-T cells prepared with rAAV6 showed evident anti-tumor ability both in vitro and in vivo. Our findings established an efficient method for the AAV transduction of T cells and would provide an alternative way for the preparation of CAR-T cells.

Enhanced Gold Nanoparticle aggregation with cancer cell DNA for improved radiation therapy and immune response in cancer treatment

In the United States, cancer is the second leading cause of death after heart disease and hundreds of thousands of people pass away each year due to this deadly disease. Many treatments for cancer are nonselective and do not provide long‐lasting immunity in case of recurrence. Gold nanoparticles can provide a more targeted radiation therapy and better effectiveness through heat generation and formation of reactive oxygen species via hydrolysis reaction which can cause DNA damage in the tumor. Unfortunately, gold particles have low photothermal transduction efficiency and must be aggregated to improve performance. To achieve this, positively charged gold nanoparticles were aggregated with MDA MB 231 cancer cell DNA to increase gold nanoparticle aggregation, to elicit higher photothermal efficiency, and to stimulate an immune response. In vitro analysis demonstrated DNA size and concentration dependent gold nanoparticle aggregation, which varied little in the pH range of 5.5 to 8.6. Furthermore, these aggregates elicited an immune response via delivery of DNA for direct transfection of antigen presenting cells. Tumor necrosis factor alpha released from mouse RAW macrophages increased in the presence of the cancer cell DNA aggregated gold particles. This work suggests that the promise of combination therapy using cancer cell DNA not only can improve photothermal therapy of gold nanoparticles, but also can elicit an immune response in cancer treatment.