Vector DNA Research Articles

Antibiotic-Free Nanoplasmids as Promising Alternatives for Conventional DNA Vectors.

DNA vaccines with their extraordinary properties are the best choice as vectors for subunit vaccines but are not in compliance with safety regulations, mainly because of the antibiotic resistance genes on their backbone. New generations of plasmids with minimum bacterial backbones are now developed as promising alternatives to pass the safety rules and be replaced for conventional plasmids. Here we have compared the nanoplasmid (with RNA-out selection system and professional HTLV-1 containing promoter) and the conventionally used pcDNA plasmid, as regards the transfection efficiency. The EGFP gene was cloned in both pcDNA-3.1+ and NTC9385R-MSC and transfected into COS-7 cells for expression evaluation by flow cytometry. Meanwhile, qPCR was used to analyze the EGFP mRNA copy numbers. It was concluded that the nanoplasmid, with its extraordinary properties, can be a tempting alternative to conventional pcDNA in equal or equimolar concentrations for vaccine design. These promising results can put DNA vaccines back into focus, especially regarding diseases controlled by robust cellular immune responses.

Intrabiliary infusion of naked DNA vectors targets periportal hepatocytes in mice

Hydrodynamic tail vein injection (HTV) is the "gold standard" for delivering naked DNA vectors to mouse liver, thereby transfecting predominately perivenous hepatocytes. While HTV corrects metabolic liver defects such as phenylketonuria or cystathionine β-synthase deficiency, correction of spf ash mice with ornithine transcarbamylase (OTC) deficiency was not possible despite overexpression in the liver, as the OTC enzyme is primarily expressed in periportal hepatocytes. To target periportal hepatocytes, we established hydrodynamic retrograde intrabiliary injection (HRII) in mice and optimized minicircle (MC) vector delivery using luciferase as a marker gene. HRII resulted in a transfection efficiency below 1%, 100-fold lower than HTV. While HRII induced minimal liver toxicity compared with HTV, overexpression of luciferase by both methods, but not of a natural liver-specific enzyme, elicited an immune response that led to the elimination of luciferase expression. Further testing of MC vectors delivered via HRII in spf ash mice did not result in sufficient therapeutic efficacy and needs further optimization and/or selection of the corrected cells. This study reveals that luciferase expression is toxic for the liver. Furthermore, physical delivery of MC vectors via the bile duct has thepotential to treat defects restricted to periportal hepatocytes, which opens new doors for non-viral liver-directed gene therapy.

Cytosolic dsRNA sensors as regulators of transgene expression from AAV vectors in human iPSC-derived cardiomyocytes

Abstract Background Adeno-associated viral vectors (AAVs) are safe and efficient tools for delivery of therapeutic genes in numerous applications. Although they elicit only limited immune response in host cells, a recent study has demonstrated an association between decreasing transgene expression in the liver and induction of the immune response to cytoplasmic dsRNA. While AAVs are DNA vectors, it appears that their unique genome structure allows the generation of dsRNA in transduced cells. Since the expression cassette is flanked by inverted terminal repeats (ITRs), which can function as bidirectional promoters, assembly of sense and antisense transcripts can potentially result in formation of dsRNA structures, especially in cells exhibiting high transgene expression. Purpose In this study, we explored the mechanism of dsRNA recognition in cardiomyocytes, to unravel its role in the regulation of transgene expression from AAV vectors. Methods For this purpose, we utilised human iPSC-derived cardiomyocytes transduced with self-complementary AAV6 or AAV9 vectors encoding GFP. Since we focused on the long-term effects of transgene expression, all the analyses were performed 7–9 days after AAV transduction. Results We confirmed that ITR serves as a functional promoter in the cells, capable of maintaining approximately half of the fluorescence signal compared to the CMV promoter. Long-term transgene expression was associated with elevated expression of dsRNA sensors (MDA5, RIG-I, LGP2 and TLR3) and the appearance of small foci of dsRNA in the perinuclear region. Unlike an artificial ligand for dsRNA sensors, poly (I:C), dsRNA in AAV-transduced cells did not interact with the LGP2 protein. To induce dsRNA recognition, we stimulated the cells with IFNβ for 1 week, starting from day 1 after transduction. We found that transgene expression is significantly hampered in such conditions. Furthermore, IFNβ induced the direct interaction of dsRNA with the OAS1 protein in transduced cells. During viral infections, OAS1 and its effector nuclease RNAse L promote the degradation of mRNA to restrict the production of viral proteins. Since RNAse L is not specific for particular transcripts, we hypothesize that activation of the OAS1 pathway in transduced cells is responsible for downregulation of transgene expression. While IFNβ induced OAS1-dsRNA recognition, we did not observe such an event in cells transduced with AAV and maintained under standard conditions. In turn, AAV transduction resulted in a reduction in OAS1 expression at the mRNA and protein level, suggesting a possible reason for tolerance to dsRNA in transduced cardiomyocytes. Conclusions Our data present a novel mechanism of regulation of transgene expression in AAV-transduced cells involving OAS1–RNAse L pathway and provide deeper insight into the general signal transduction routes activated in response to cytosolic dsRNA in human iPSC-derived cardiomyocytes. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Science Centre - PRELUDUM grant 2019/33/N/NZ1/03066Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology - grant for young scientists MNS 6/2020

Minicircle Delivery to the Neural Retina as a Gene Therapy Approach.

Non-viral gene therapy has the potential to overcome several shortcomings in viral vector-based therapeutics. Methods of in vivo plasmid delivery have developed over recent years to increase the efficiency of non-viral gene transfer, yet further improvements still need to be made to improve their translational capacity. Gene therapy advances for inherited retinal disease have been particularly prominent over the recent decade but overcoming physical and physiological barriers present in the eye remains a key obstacle in the field of non-viral ocular drug delivery. Minicircles are circular double-stranded DNA vectors that contain expression cassettes devoid of bacterial DNA, thereby limiting the risks of innate immune responses induced by such elements. To date, they have not been extensively used in pre-clinical studies yet remain a viable vector option for the treatment of inherited retinal disease. Here, we explore the potential of minicircle DNA delivery to the neural retina as a gene therapy approach. We consider the advantages of minicircles as gene therapy vectors as well as review the challenges involved in optimising their delivery to the neural retina.

P.130 RGX-202: An investigational AAV8 gene therapy coding for a novel microdystrophin as a treatment for Duchenne muscular dystrophy

RGX-202 is an investigational one-time gene therapy for the treatment of Duchenne muscular dystrophy (Duchenne) that utilizes a NAV AAV8 vector, a muscle specific promoter (Spc5-12), and an optimized microdystrophin transgene. The microdystrophin coding sequence involves codon optimization, depletion of CpG dinucleotides, and an extended coding region for the C-Terminal (CT) domain found in naturally occurring dystrophin. Preclinical studies in mdx mice demonstrated a dose proportional increase of RGX-202 vector DNA and microdystrophin in skeletal and heart muscle and restoration of the dystrophin-associated complex (DAPC) at the sarcolemma, indicating the potential benefit of the CT domain. Moreover, after a single intravenous (IV) injection of RGX-202, mdx mice showed significant improvements in dystrophic muscle pathology (muscle lesions and fibrosis) and function (in vitro force, and automated gait analysis) at doses > 1e14 GC/kg. A Phase I/II trial, AFFINITY DUCHENNE, is a multicenter, open-label dose escalation study to evaluate the safety, tolerability and clinical efficacy of a one-time IV dose of RGX-202 in patients with Duchenne. Six ambulatory, pediatric patients (ages 4 to 11 years old) with Duchenne are expected to enroll in 2 cohorts at doses of 1x10¹⁴ genome copies (GC)/kg body weight (n=3) and 2x10¹⁴ GC/kg body weight (n=3). An expansion may allow for up to 6 additional participants to be enrolled at each dose for a total of up to 9 participants at each dose and 18 participants in the study. Additional inclusion criteria are based on dystrophin gene mutations in exons 18-58, a functional assessment, and AAV seronegative status. Endpoints include safety, immunogenicity assessments, RGX-202 microdystrophin levels in muscle, functional assessments (North Star Ambulatory Assessment and timed function tests), and muscle preservation (MRI).

Towards the development of an epitope-focused vaccine for SARS-CoV-2

The rapid spread of COVID-19 on all continents and the mortality induced by SARS-CoV-2 virus, the cause of the pandemic coronavirus disease 2019 (COVID-19) has motivated an unprecedented effort for vaccine development. Inactivated viruses as well as vaccines focused on the partial or total sequence of the Spike protein using different novel platforms such us RNA, DNA, proteins, and non-replicating viral vectors have been developed. The high global need for vaccines, now and in the future, and the emergence of new variants of concern still requires development of accessible vaccines that can be adapted according to the most prevalent variants in the respective regions.Here, we describe the immunogenic properties of a group of theoretically predicted RBD peptides to be used as the first step towards the development of an effective, safe and low-cost epitope-focused vaccine. One of the tested peptides named P5, proved to be safe and immunogenic. Subcutaneous administration of the peptide, formulated with alumina, induced high levels of specific IgG antibodies in mice and hamsters, as well as an increase of IFN-γ expression by CD8+ T cells in C57 and BALB/c mice upon in vitro stimulation with P5. Neutralizing titers of anti-P5 antibodies, however, were disappointingly low, a deficiency that we will attempt to resolve by the inclusion of additional immunogenic epitopes to P5. The safety and immunogenicity data reported in this study support the use of this peptide as a starting point for the design of an epitope restricted vaccine.

Versatile seamless DNA vector production in E. coli using enhanced phage lambda integrase.

Seamless DNA vectors derived from bacterial plasmids are devoid of bacterial genetic elements and represent attractive alternatives for biomedical applications including DNA vaccines. Larger scale production of seamless vectors employs engineered Escherichia coli strains in order to enable tightly regulated expression of site-specific DNA recombinases which precisely delete unwanted sequences from bacterial plasmids. As a novel component of a developing lambda integrase genome editing platform, we describe here strain MG1655-ISC as a means to easily produce different scales of seamless vectors, ranging in size from a few hundred base pairs to more than ten kilo base pairs. Since we employed an engineered lambda integrase that is able to efficiently recombine pairs of DNA crossover sites that differ in sequence, the resulting seamless vectors will be useful for subsequent genome editing in higher eukaryotes to accommodate variations in target site sequences. Future inclusion of single cognate sites for other genome targeting systems could enable modularity. These features, together with the demonstrated simplicity of in vivo seamless vector production, add to their utility in the biomedical space.

Temoneira-1 β-lactamase is not a metalloenzyme, but its native metal ion binding sites allow for purification by immobilized metal ion affinity chromatography

β-lactamases protect bacteria from β-lactam antibiotics. Temoneira (TEM) is a class A serine β-lactamase and its coding sequence is designed into DNA vectors, such as pET-21a (+), to provide antibiotic resistance. TEM-1 β-lactamase was overexpressed efficiently from this vector upon inducing protein expression by IPTG in BL21(DE3) cells. Immobilized metal ion affinity chromatography (IMAC) was used based on the three native putative metal ion binding sites of TEM-1 β-lactamase, each consisting of a pair of histidine sidechains. Elution was achieved at low concentrations of imidazole (∼15–200 mM). Two steps of IMAC and a subsequent anion exchange purification produced highly pure TEM-1 β-lactamase with a yield of 1.9 mg/g of wet bacterial pellet weight. Mass spectrometry revealed that the mature form of β-lactamase (without the signal sequence) was obtained. The secondary structure composition, calculated from the circular dichroism spectrum, showed that the target protein was folded similar to the published crystal structure. Ni(II) binding to the enzyme was also investigated. Increasing amounts of Ni(II) ions had only a small effect on the protein structure. Mass spectrometry detected up to three bound metal ions at 10:1 Ni(II):protein molar ratio, but the major peak was assigned to the monometallated β-lactamase indicating the presence of a paramount metal ion binding site formed by the H151/H156 pair.

What’s new and what’s next for gene therapy in Pompe disease?

Introduction Pompe disease is an autosomal recessive disorder caused by a deficiency of acid-⍺-glucosidase (GAA), an enzyme responsible for hydrolyzing lysosomal glycogen. A lack of GAA leads to accumulation of glycogen in the lysosomes of cardiac, skeletal, and smooth muscle cells, as well as in the central and peripheral nervous system. Enzyme replacement therapy has been the standard of care for 15 years and slows disease progression, particularly in the heart, and improves survival. However, there are limitations of ERT success, which gene therapy can overcome. Areas covered Gene therapy offers several advantages including prolonged and consistent GAA expression and correction of skeletal muscle as well as the critical CNS pathology. We provide a systematic review of the preclinical and clinical outcomes of adeno-associated viral mediated gene therapy and alternative gene therapy strategies, highlighting what has been successful. Expert opinion Although the preclinical and clinical studies so far have been promising, barriers exist that need to be addressed in gene therapy for Pompe disease. New strategies including novel capsids for better targeting, optimized DNA vectors, and adjuctive therapies will allow for a lower dose, and ameliorate the immune response.

Recombinant Viral Vectors for Therapeutic Programming of Tumour Microenvironment: Advantages and Limitations.

Viral vectors have been widely investigated as tools for cancer immunotherapy. Although many preclinical studies demonstrate significant virus-mediated tumour inhibition in synergy with immune checkpoint molecules and other drugs, the clinical success of viral vector applications in cancer therapy currently is limited. A number of challenges have to be solved to translate promising vectors to clinics. One of the key elements of successful virus-based cancer immunotherapy is the understanding of the tumour immune state and the development of vectors to modify the immunosuppressive tumour microenvironment (TME). Tumour-associated immune cells, as the main component of TME, support tumour progression through multiple pathways inducing resistance to treatment and promoting cancer cell escape mechanisms. In this review, we consider DNA and RNA virus vectors delivering immunomodulatory genes (cytokines, chemokines, co-stimulatory molecules, antibodies, etc.) and discuss how these viruses break an immunosuppressive cell development and switch TME to an immune-responsive “hot” state. We highlight the advantages and limitations of virus vectors for targeted therapeutic programming of tumour immune cell populations and tumour stroma, and propose future steps to establish viral vectors as a standard, efficient, safe, and non-toxic cancer immunotherapy approach that can complement other promising treatment strategies, e.g., checkpoint inhibitors, CAR-T, and advanced chemotherapeutics.

Design, Development, and Testing of a Device for Gene Electrotransfer to Skin Cells In Vivo.

Gene electrotransfer (GET) is considered one of the most efficient, safe, reproducible, and cost-effective methods of gene therapy, in which a gene is delivered to the cells in the form of a plasmid DNA vector by a method known as electroporation. To achieve successful electroporation, cells must be exposed to sufficiently high electric fields generated by short-duration, high-voltage electrical pulses that result in a temporary increase in plasma membrane permeability. The electrical pulses are generated by pulse generators (electroporators) and delivered to the cells via electrodes (applicators). However, there is a lack of standardized pulse delivery protocols as well as certified clinical pulse generators and applicators for gene delivery. In this paper, the development of a new pulse generator, applicator, and pulse delivery protocol for GET to skin cells is presented. A numerical model of electroporated skin developed and tested for two electrode configurations and two different pulse delivery protocols is also presented. An alternative pulse delivery protocol was proposed. The developed pulse generator, applicator, and the proposed pulse delivery protocol were then used in vivo for GET to skin cells in mice. The results showed high efficiency of the proposed pulse delivery protocol for the purpose of GET in mouse skin cells. Specifically, electroporation with the developed pulse generator, applicator, and proposed pulse delivery protocol resulted in higher gene expression in skin cells compared to the currently used pulse generator, applicator, and pulse delivery protocol.

A Pan-RNase Inhibitor Enabling CRISPR-mRNA Platforms for Engineering of Primary Human Monocytes.

Monocytes and their downstream effectors are critical components of the innate immune system. Monocytes are equipped with chemokine receptors, allowing them to migrate to various tissues, where they can differentiate into macrophage and dendritic cell subsets and participate in tissue homeostasis, infection, autoimmune disease, and cancer. Enabling genome engineering in monocytes and their effector cells will facilitate a myriad of applications for basic and translational research. Here, we demonstrate that CRISPR-Cas9 RNPs can be used for efficient gene knockout in primary human monocytes. In addition, we demonstrate that intracellular RNases are likely responsible for poor and heterogenous mRNA expression as incorporation of pan-RNase inhibitor allows efficient genome engineering following mRNA-based delivery of Cas9 and base editor enzymes. Moreover, we demonstrate that CRISPR-Cas9 combined with an rAAV vector DNA donor template mediates site-specific insertion and expression of a transgene in primary human monocytes. Finally, we demonstrate that SIRPa knock-out monocyte-derived macrophages have enhanced activity against cancer cells, highlighting the potential for application in cellular immunotherapies.

Recent Progress in the Discovery and Development of Monoclonal Antibodies against Viral Infections.

Monoclonal antibodies (mAbs), the new revolutionary class of medications, are fast becoming tools against various diseases thanks to a unique structure and function that allow them to bind highly specific targets or receptors. These specialized proteins can be produced in large quantities via the hybridoma technique introduced in 1975 or by means of modern technologies. Additional methods have been developed to generate mAbs with new biological properties such as humanized, chimeric, or murine. The inclusion of mAbs in therapeutic regimens is a major medical advance and will hopefully lead to significant improvements in infectious disease management. Since the first therapeutic mAb, muromonab-CD3, was approved by the U.S. Food and Drug Administration (FDA) in 1986, the list of approved mAbs and their clinical indications and applications have been proliferating. New technologies have been developed to modify the structure of mAbs, thereby increasing efficacy and improving delivery routes. Gene delivery technologies, such as non-viral synthetic plasmid DNA and messenger RNA vectors (DMabs or mRNA-encoded mAbs), built to express tailored mAb genes, might help overcome some of the challenges of mAb therapy, including production restrictions, cold-chain storage, transportation requirements, and expensive manufacturing and distribution processes. This paper reviews some of the recent developments in mAb discovery against viral infections and illustrates how mAbs can help to combat viral diseases and outbreaks.

Nonviral mcDNA-mediated bispecific CAR T cells kill tumor cells in an experimental mouse model of hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is one of the most common cancers worldwide and the adoptive immunotherapy of which is worth studying. CD133, a kind of cancer stem cell (CSC) antigen, together with glypican-3 (GPC3) has been proved to be highly expressed in HCC cells and both of them are used as targets to generate chimeric antigen receptor (CAR) T cells. But there are limitations like “off-target” toxicity, low transfection efficacy and weak antitumor ability in CAR T cells treatment.MethodsThe peripheral blood was acquired from healthy donors and T cells were separated by density-gradient centrifugation. We used an electroporation system to deliver anti-CD133 and anti-GPC3 single chain Fragment variable (scFv) structures as target genes into the T cells. The cell membrane was opened by the momentary electric current effect, and the target gene was delivered into the cell by non-viral minicircle DNA (mcDNA) vector. The flow cytometry and western blot assays were used to detect whether the two scFv were simultaneously transfected and the transfection efficacy of this bispecific CAR T cell generation method. We respectively detected the in vitro and in vivo tumor-suppression efficacy of CAR T cells through the CCK-8 assays and the HCC xenograft mice models. The CoG133-CAR T cells containing both CD133 and GPC3 antigen recognition sites were the effector cells. CD133-CAR T cells and GPC3-CAR T cells were defined as single-targeted control groups, normal T and mock T cells were defined as blank control groups.ResultsThe mcDNA vector accommodated two target gene structures successfully transfected to generate bispecific CAR T cells. The detection methods on gene level and protein level confirmed that CoG133-CAR T cells had considerable transfection efficiency and exhibited both antigen-binding capacity of CD133 and GPC3. Compared to single-targeted CAR T cells or control T cells, CoG133-CAR T cells performed enhanced eliminated efficacy against CD133 and GPC3 double-positive HCC cell line in vitro and HCC xenograft mice in vivo. Hematoxylin and eosin (H&E) staining indicated no fatal “off-target” combination existed on CoG133-CAR T cells and major organs.ConclusionOur study suggests that it is with higher efficiency and more safety to prepare bispecific CAR T cells through non-viral mcDNA vectors. CoG133-CAR T cells have enhanced tumor-suppression capacity through dual antigen recognition and internal activation. It provides an innovative strategy for CAR T therapy of HCC, even solid tumors.

Early and Long-Term HIV-1 Immunogenicity Induced in Macaques by the Combined Administration of DNA, NYVAC and Env Protein-Based Vaccine Candidates: The AUP512 Study.

To control HIV infection there is a need for vaccines to induce broad, potent and long-term B and T cell immune responses. With the objective to accelerate and maintain the induction of substantial levels of HIV-1 Env-specific antibodies and, at the same time, to enhance balanced CD4 and CD8 T cell responses, we evaluated the effect of concurrent administration of MF59-adjuvanted Env protein together with DNA or NYVAC vectors at priming to establish if early administration of Env leads to early induction of antibody responses. The primary goal was to assess the immunogenicity endpoint at week 26. Secondary endpoints were (i) to determine the quality of responses with regard to RV144 correlates of protection and (ii) to explore a potential impact of two late boosts. In this study, five different prime/boost vaccination regimens were tested in rhesus macaques. Animals received priming immunizations with either NYVAC or DNA alone or in combination with Env protein, followed by NYVAC + protein or DNA + protein boosts. All regimens induced broad, polyfunctional and well-balanced CD4 and CD8 T cell responses, with DNA-primed regimens eliciting higher response rates and magnitudes than NYVAC-primed regimens. Very high plasma binding IgG titers including V1/V2 specific antibodies, modest antibody-dependent cellular cytotoxicity (ADCC) and moderate neutralization activity were observed. Of note, early administration of the MF59-adjuvanted Env protein in parallel with DNA priming leads to more rapid elicitation of humoral responses, without negatively affecting the cellular responses, while responses were rapidly boosted after repeated immunizations, indicating the induction of a robust memory response. In conclusion, our findings support the use of the Env protein component during priming in the context of an heterologous immunization regimen with a DNA and/or NYVAC vector as an optimized immunization protocol against HIV infection.

Front Cover: Advances towards Cell‐Specific Gene Transfection: A Small‐Molecule Approach Allows Order‐of‐Magnitude Selectivity (Chem. Eur. J. 43/2022)

A biotin-labelled transfection vector tightly grabs a DNA molecule with a binding unit of similar size to the label. At the same time, a cancer cell scans the label with its biotin receptor. While this will prompt the greedy cancer cell to be transfected by the DNA vector system, other cells are safe. And you can feel just as safe, if you want to use your smartphone and a scanner app in order to check out what information is hidden behind the QR code. More information can be found in the Research Article by C. Hirschhäuser et al. (DOI: 10.1002/chem.202104618).

A DNA-Free Editing Platform for Genetic Screens in Soybean via CRISPR/Cas9 Ribonucleoprotein Delivery.

CRISPR/Cas9-based ribonucleoprotein (RNP)-mediated system has the property of minimizing the effects related to the unwanted introduction of vector DNA and random integration of recombinant DNA. Here, we describe a platform based on the direct delivery of Cas9 RNPs to soybean protoplasts for genetic screens in knockout gene-edited soybean lines without the transfection of DNA vectors. The platform is based on the isolation of soybean protoplasts and delivery of Cas RNP complex. To empirically test our platform, we have chosen a model gene from the soybean genetic toolbox. We have used five different guide RNA (gRNA) sequences that targeted the constitutive pathogen response 5 (CPR5) gene associated with the growth of trichomes in soybean. In addition, efficient protoplast transformation, concentration, and ratio of Cas9 and gRNAs were optimized for soybean for the first time. Targeted mutagenesis insertion and deletion frequency and sequences were analyzed using both Sanger and targeted deep sequencing strategies. We were able to identify different mutation patterns within insertions and deletions (InDels) between + 5 nt and –30 bp and mutation frequency ranging from 4.2 to 18.1% in the GmCPR5 locus. Our results showed that DNA-free delivery of Cas9 complexes to protoplasts is a useful approach to perform early-stage genetic screens and anticipated analysis of Cas9 activity in soybeans.

Absence of lenadogene nolparvovec DNA in a brain tumor biopsy from a patient in the REVERSE clinical study, a case report

BackgroundLeber Hereditary Optic Neuropathy (LHON) is a rare, maternally-inherited mitochondrial disease that primarily affects retinal ganglion cells (RGCs) and their axons in the optic nerve, leading to irreversible, bilateral severe vision loss. Lenadogene nolparvovec gene therapy was developed as a treatment for patients with vision loss from LHON caused by the most prevalent m.11778G > A mitochondrial DNA point mutation in the MT-ND4 gene. Lenadogene nolparvovec is a replication-defective recombinant adeno-associated virus vector 2 serotype 2 (AAV2/2), encoding the human wild-type MT-ND4 protein. Lenadogene nolparvovec was administered by intravitreal injection (IVT) in LHON patients harboring the m.11778G > A ND4 mutation in a clinical development program including one phase 1/2 study (REVEAL), three phase 3 pivotal studies (REVERSE, RESCUE, REFLECT), and one long-term follow-up study (RESTORE, the follow-up of REVERSE and RESCUE patients).Case presentationA 67-year-old woman with MT-ND4 LHON, included in the REVERSE clinical study, received a unilateral IVT of lenadogene nolparvovec in the right eye and a sham injection in the left eye in May 2016, 11.4 months and 8.8 months after vision loss in her right and left eyes, respectively. The patient had a normal brain magnetic resonance imaging with contrast at the time of diagnosis of LHON. Two years after treatment administration, BCVA had improved in both eyes. The product was well tolerated with mild and resolutive anterior chamber inflammation in the treated eye. In May 2019, the patient was diagnosed with a right temporal lobe glioblastoma, IDH-wildtype, World Health Organization grade 4, based on histological analysis of a tumor excision. The brain tumor was assessed for the presence of vector DNA by using a sensitive validated qPCR assay targeting the ND4 sequence of the vector.ConclusionND4 DNA was not detected (below 15.625 copies/μg of genomic DNA) in DNA extracted from the brain tumor, while a housekeeping gene DNA was detected at high levels. Taken together, this data shows the absence of detection of lenadogene nolparvovec in a brain tumor (glioblastoma) of a treated patient in the REVERSE clinical trial 3 years after gene therapy administration, supporting the long-term favorable safety of lenadogene nolparvovec.

Activation of Anti-SARS-CoV-2 Human CTLs by Extracellular Vesicles Engineered with the N Viral Protein.

We propose an innovative anti-SARS-CoV-2 immune strategy based on extracellular vesicles (EVs) inducing an anti-SARS-CoV-2 N CD8+ T cytotoxic lymphocyte (CTL) immune response. We previously reported that the SARS-CoV-2 N protein can be uploaded at high levels in EVs upon fusion with Nefmut, i.e., a biologically inactive HIV-1 Nef mutant incorporating into EVs at quite high levels. Here, we analyze the immunogenic properties in human cells of EVs engineered with SARS-CoV-2 N fused at the C-terminus of either Nefmut or a deletion mutant of Nefmut referred to as NefmutPL. The analysis of in vitro-produced EVs has supported the uploading of N protein when fused with truncated Nefmut. Mice injected with DNA vectors expressed each fusion protein developed robust SARS-CoV-2 N-specific CD8+ T cell immune responses. When ex vivo human dendritic cells were challenged with EVs engineered with either fusion products, the induction of a robust N-specific CTL activity, as evaluated by both CD107a and trogocytosis assays, was observed. Through these data we achieved the proof-of-principle that engineered EVs can be instrumental to elicit anti-SARS-CoV-2 CTL immune response in human cells. This achievement represents a mandatory step towards the upcoming experimentations in pre-clinical models focused on intranasal administration of N-engineered EVs.

Abstract 3564: Vaccination using different platforms encoding HER2-LAMP with heterologous boosting enhances adaptive HER2-specific immunity to enable potent anti-tumor responses

Abstract Background: Immune checkpoint blockade (ICB) antibodies have demonstrated that effective induction of T cell responses can elicit tumor regression of different metastatic cancers, although these responses are restricted to certain individuals with specific types of cancer. To enhance these responses, there has been renewed emphasis in developing different types of cancer-specific vaccines to stimulate and direct T cell and B cell immunity to important oncologic targets, such as the oncogene human epidermal growth factor receptor 2 (HER2), which is expressed in ~20% of breast cancers (BC). In our previous work we used a DNA plasmid vaccine targeting HER2, which utilized a lysosome-associated membrane protein 1 (LAMP) domain to traffic HER2 antigen to endolysosomal compartments. We demonstrated that this approach was more effective at eliciting antigen specific CD4+ and CD8+ T cell responses, and provided a significant but limited survival benefit in an endogenous mouse model of HER2+ BC compared to a HER2-wild type vaccine. Methods: In our current study, we explored the use of this modified antigen construct using different vaccine modalities, including DNA plasmid, adenoviral (Ad), modified vaccinia ankara (MVA), and self-replicating RNA vaccines to identify effective vaccine platforms to elicit HER2-specific immunity and anti-tumor immunity. Additionally, we have tested the combination of these modalities in homologous and heterologous vaccine boosting regimens to determine an optimal strategy to elicit anti-tumor immunity in vivo. Results: In our studies, plasmid and MVA vectors elicited suboptimal immune priming responses, in comparison to Ad and self-replicating RNA vectors that elicit significant HER2-specific T and B cell responses upon vaccination. While timing of boosting is critical, our results reflect that certain platforms (e.g., plasmid DNA vectors) are capable of homologous boosting capacity, while others (e.g., Ad vectors) elicit more robust T and B cell responses to capsid epitopes, limiting homologous boosting capacity for HER2-specific immunity. Significantly, using an endogenous model of metastatic HER2+ BC, we have found that heterologous vaccination with different HER2-LAMP targeted vectors can elicit significantly augmented HER2-specific T and B cell responses that translated into more effective anti-tumor immunity. This immunity proved capable of eliciting tumor regression in ~80% of vaccinated mice, compared to 0% using homologous HER2-WT plasmid vaccination, 30% using homologous HER2-LAMP plasmid vaccination, or 50% with HER2-WT heterologous vaccination. Conclusions: These data demonstrate the potential of utilizing heterologous vaccine platforms encoding LAMP-based endolysosomal trafficking vaccines to elicit HER2-specific immunity and effect anti-tumor responses. Citation Format: Robert D. Marek, Alan Chen, Renhuan Xu, Junping Wei, Tao Wang, Xiao Yang, Gangjun Lei, Teri Heiland, Zachary C. Hartman. Vaccination using different platforms encoding HER2-LAMP with heterologous boosting enhances adaptive HER2-specific immunity to enable potent anti-tumor responses [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 3564.