Viral Vector Systems Research Articles

Efficient virus-induced gene silencing in Primulina using two virus vector systems

Virus-induced gene silencing (VIGS) is a post-transcriptional gene silencing method and an effective technique for analyzing plant gene function. However, to date there have been no reports on the establishment of a VIGS system in Primulina, which includes over 200 species of plants, most of which have high ornamental value. In this study, we successfully established a VIGS system in P. 'Alfonso' and P. carinata using TRV vector system with phytoene desaturase (PDS) as marker gene. After being treated with pTRV2-PDS vector, both P. 'Alfonso' and P. carinata plants showed a pronounced photobleaching phenotype. The system was further optimized by exploring different infection conditions. The optimized TRV-based VIGS system was as follows: Agrobacterium OD600 = 0.5, leaf vacuum infiltration, and the silencing efficiency of the system can reach 75 %. Chlorata42 was used as another marker gene and further verified the establishment of TRV VIGS system in P. 'Alfonso'. In addition, the CaLCuV-based VIGS system was also established with PDS as marker gene in P. 'Alfonso'. The establishment of the TRV-based and CaLCuV-based VIGS systems provided a breakthrough in the validation methods for identifying gene function in Primulina genus.

AAV-mouse DNase I sustains long-term DNase I expression invivo and suppresses breast cancer metastasis.

Neutrophil extracellular traps (NETs) have been implicated in the pathology of various inflammatory conditions. In cancer, NETs have been demonstrated to induce systemic inflammation, impair peripheral vessel and organ function and promote metastasis. Here we show that the plasma level of NETs is significantly higher in patients with metastatic breast cancer compared to those with local disease, or those that were considered cured at a 5-year follow-up, confirming NETs as interesting therapeutic targets in metastatic breast cancer. Administration of DNase I is one strategy to eliminate NETs but long-term treatment requires repeated injections and species-specific versions of the enzyme. To enhance administration and therapeutic efficacy, we have developed an adeno-associated virus (AAV) vector system for delivery of murine DNase I and addressed its potential to counteract cancer-associated pathology in the murine MMTV-PyMT model for metastatic mammary carcinoma. The AAV vector is comprised of capsid KP1 and an expression cassette encoding hyperactive murine DNase I (AAV-mDNase I) under the control of a liver-specific promotor. This AAV-mDNase I vector could support elevated expression and serum activity of murine DNase I over at least 8 months. Neutrophil Gelatinase-Associated Lipocalin (NGAL), a biomarker for kidney hypoperfusion that is upregulated in urine from MMTV-PyMT mice, was suppressed in mice receiving AAV-mDNase I compared to an AAV-null control group. Furthermore, the proportion of mice that developed lung metastasis was reduced in the AAV-mDNase I group. Altogether, our data indicate that AAV-mDNase I has the potential to reduce cancer-associated impairment of renal function and development of metastasis. We conclude that AAV-mDNase I could represent a promising therapeutic strategy in metastatic breast cancer.

Comparing Viral Vectors and Fate Mapping Approaches for Astrocyte-to-Neuron Reprogramming in the Injured Mouse Cerebral Cortex.

Direct neuronal reprogramming is a promising approach to replace neurons lost due to disease via the conversion of endogenous glia reacting to brain injury into neurons. However, it is essential to demonstrate that the newly generated neurons originate from glial cells and/or show that they are not pre-existing endogenous neurons. Here, we use controls for both requirements while comparing two viral vector systems (Mo-MLVs and AAVs) for the expression of the same neurogenic factor, the phosphorylation-resistant form of Neurogenin2. Our results show that Mo-MLVs targeting proliferating glial cells after traumatic brain injury reliably convert astrocytes into neurons, as assessed by genetic fate mapping of astrocytes. Conversely, expressing the same neurogenic factor in a flexed AAV system results in artefactual labelling of endogenous neurons fatemapped by birthdating in development that are negative for the genetic fate mapping marker induced in astrocytes. These results are further corroborated by chronic live in vivo imaging. Taken together, the phosphorylation-resistant form of Neurogenin2 is more efficient in reprogramming reactive glia into neurons than its wildtype counterpart in vivo using retroviral vectors (Mo-MLVs) targeting proliferating glia. Conversely, AAV-mediated expression generates artefacts and is not sufficient to achieve fate conversion.

Prevalence and Diversity of Aphid-Vectored Yellow Dwarf Viruses in Oregon Perennial Grass Seed Crops

Oregon produces 70% of the world's cool-season grass seed, many species of which are susceptible to yellow dwarf viruses (YDVs). This virus complex has numerous defined virus species vectored by multiple aphid species. The diversity of YDVs associated with Oregon grass seed production has not been determined and is needed to provide insight to develop a risk prediction framework and improve management guidelines to mitigate disease severity. Commercial grass seed fields ( n = 57) across the Willamette Valley and central and eastern Oregon were surveyed across 2021 and 2022 to determine the incidence and diversity of YDVs associated with crop species (perennial ryegrass, tall fescue, and Kentucky bluegrass) and the abundance and incidence of YDVs associated with aphid vectors. Virus incidence and diversity were determined using multiple previously published endpoint reverse-transcriptase multiplexes specific for YDV genera and species. Across commercial grass seed crop hosts and growing seasons, 82% of fields had at least one plant sample detection of a luteovirus-type YDV, and 65% had at least one detection of a polerovirus-type YDV. Both perennial ryegrass and tall fescue crops hosted diverse YDV populations, which was especially apparent during the spring sampling season, when similarly diverse YDV communities were found associated with alate aphids. Only polerovirus-type YDVs were detected in Kentucky bluegrass, reflecting a variable landscape compared to Oregon's western Willamette Valley. This study provides a current understanding of the spatial composition and diversity of the aphid–YDV virus–vector system in grasses grown for seed in Oregon. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

Molecular Methods for the Simultaneous Detection of Tomato Fruit Blotch Virus and Identification of Tomato Russet Mite, a New Potential Virus–Vector System Threatening Solanaceous Crops Worldwide

Tomato fruit blotch virus (ToFBV) (Blunervirus solani, family Kitaviridae) was firstly identified in Italy in 2018 in tomato plants that showed the uneven, blotchy ripening and dimpling of fruits. Subsequent High-Throughput Sequencing (HTS) analysis allowed ToFBV to be identified in samples collected in Australia, Brazil, and several European countries, and its presence in tomato crops was dated back to 2012. In 2023, the virus was found to be associated with two outbreaks in Italy and Belgium, and it was included in the EPPO Alert list as a potential new threat for tomato fruit production. Many epidemiologic features of ToFBV need to be still clarified, including transmission. Aculops lycopersici Massee (Acariformes: Eriophyoidea), the tomato russet mite (TRM), is a likely candidate vector, since high population densities were found in most of the ToFBV-infected tomato cultivations worldwide. Real-time RT-PCR tests for ToFBV detection and TRM identification were developed, also as a duplex assay. The optimized tests were then transferred to an RT-ddPCR assay and validated according to the EPPO Standard PM 7/98 (5). Such sensitive, reliable, and validated tests provide an important diagnostic tool in view of the probable threat posed by this virus–vector system to solanaceous crops worldwide and can contribute to epidemiological studies by simplifying the efficiency of research. To our knowledge, these are the first molecular methods developed for the simultaneous detection and identification of ToFBV and TRM.

CRISPR-Cas12a for Highly Efficient and Marker-Free Targeted Integration in Human Pluripotent Stem Cells.

The CRISPR-Cas12a platform has attracted interest in the genome editing community because the prototypical Acidaminococcus Cas12a generates a staggered DNA double-strand break upon binding to an AT-rich protospacer-adjacent motif (PAM, 5'-TTTV). The broad application of the platform in primary human cells was enabled by the development of an engineered version of the natural Cas12a protein, called Cas12a Ultra. In this study, we confirmed that CRISPR-Cas12a Ultra ribonucleoprotein complexes enabled allelic gene disruption frequencies of over 90% at multiple target sites in human T cells, hematopoietic stem and progenitor cells (HSPCs), and induced pluripotent stem cells (iPSCs). In addition, we demonstrated, for the first time, the efficient knock-in potential of the platform in human iPSCs and achieved targeted integration of a GFP marker gene into the AAVS1 safe harbor site and a CSF2RA super-exon into CSF2RA in up to 90% of alleles without selection. Clonal analysis revealed bi-allelic integration in >50% of the screened iPSC clones without compromising their pluripotency and genomic integrity. Thus, in combination with the adeno-associated virus vector system, CRISPR-Cas12a Ultra provides a highly efficient genome editing platform for performing targeted knock-ins in human iPSCs.

DNA-Based Nonviral Gene Therapy─Challenging but Promising.

Over the past decades, significant progress has been made in utilizing nucleic acids, including DNA and RNA molecules, for therapeutic purposes. For DNA molecules, although various DNA delivery systems have been established, viral vector systems are the go-to choice for large-scale commercial applications. However, viral systems have certain disadvantages such as immune response, limited payload capacity, insertional mutagenesis and pre-existing immunity. In contrast, nonviral systems are less immunogenic, not size limited, safer, and easier for manufacturing compared with viral systems. What's more, nonviral DNA vectors have demonstrated their capacity to mediate specific protein expression in vivo for diverse therapeutic objectives containing a wide range of diseases such as cancer, rare diseases, neurodegenerative diseases, and infectious diseases, yielding promising therapeutic outcomes. However, exogenous plasmid DNA is prone to degrade and has poor immunogenicity in vivo. Thus, various strategies have been developed: (i) designing novel plasmids with special structures, (ii) optimizing plasmid sequences for higher expression, and (iii) developing more efficient nonviral DNA delivery systems. Based on these strategies, many interesting clinical results have been reported. This Review discusses the development of DNA-based nonviral gene therapy, including novel plasmids, nonviral delivery systems, clinical advances, and prospects. These developments hold great potential for enhancing the efficacy and safety of nonviral gene therapy and expanding its applications in the treatment of various diseases.

Continuous directed evolution of a compact CjCas9 variant with broad PAM compatibility.

CRISPR-Cas9 genome engineering is a powerful technology for correcting genetic diseases. However, the targeting range of Cas9 proteins is limited by their requirement for a protospacer adjacent motif (PAM), and in vivo delivery is challenging due to their large size. Here, we use phage-assisted continuous directed evolution to broaden the PAM compatibility of Campylobacter jejuni Cas9 (CjCas9), the smallest Cas9 ortholog characterized to date. The identified variant, termed evoCjCas9, primarily recognizes N4AH and N5HA PAM sequences, which occur tenfold more frequently in the genome than the canonical N3VRYAC PAM site. Moreover, evoCjCas9 exhibits higher nuclease activity than wild-type CjCas9 on canonical PAMs, with editing rates comparable to commonly used PAM-relaxed SpCas9 variants. Combined with deaminases or reverse transcriptases, evoCjCas9 enables robust base and prime editing, with the small size of evoCjCas9 base editors allowing for tissue-specific installation of A-to-G or C-to-T transition mutations from single adeno-associated virus vector systems.

A geminivirus crosses the monocot-dicot boundary and acts as a viral vector for gene silencing and genome editing

IntroductionMembers of the family Geminiviridae have been reported to infect either a monocot plant or a dicot plant, but not both. This study reports a geminivirus, Wheat Dwarf India Virus (WDIV), first identified in wheat, that is capable of infecting both monocot and dicot plants and acting as a viral vector. ObjectivesThis study was aimed at developing a broad host range viral vector system for reverse genetics and genome editing. MethodsHere we used a wheat isolate of WDIV and Ageratum yellow leaf curl betasatellite (AYLCB) for infectivity assays and vector development. We performed Agrobacterium-mediated inoculation of WDIV and AYLCB in wheat, oat, barley, corn, soybean, and tobacco. To examine the potential of WDIV to act as a viral vector, we modified the WDIV genome and cloned DNA fragments of the phytoene desaturase (PDS) genes from wheat and tobacco, separately. For gene editing experiments, tobacco lines expressing Cas9 were infiltrated with a WDIV-based vector carrying gRNA targeting the PDS gene. ResultsAbout 80 to 90% of plants inoculated with infectious clones of WDIV alone or WDIV together with AYLCB showed mild symptoms, whereas some plants showed more prominent symptoms. WDIV and AYLCB were detected in the systemically infected leaves of all the plant species. Furthermore, the inoculation of the WDIV vector carrying PDS fragments induced silencing of the PDS gene in both wheat and tobacco plants. We also observed high-efficiency genome editing in the Cas9-expressing tobacco plants that were inoculated with WDIV vector-carrying gRNA. ConclusionDetection of WDIV in naturally infected wheat, barley, and sugarcane in the field and its ability to systemically infect wheat, oat, barley, corn, soybean, and tobacco under laboratory conditions, provides compelling evidence that WDIV is the first geminivirus identified with the capability of infecting both monocot and dicot plant species. The wide host range of WDIV can be exploited for developing a single vector system for high-throughput genome editing in many plant species.

Generation and Maintenance of Primate Induced Pluripotent Stem Cells Derived from Urine.

Cross-species approaches studying primate pluripotent stem cells and their derivatives are crucial to better understand the molecular and cellular mechanisms of disease, development, and evolution. To make primate induced pluripotent stem cells (iPSCs) more accessible, this paper presents a non-invasive method to generate human and non-human primate iPSCs from urine-derived cells, and their maintenance using a feeder-free culturing method. The urine can be sampled from a non-sterile environment (e.g., the cage of the animal) and treated with a broad-spectrum antibiotic cocktail during primary cell culture to reduce contamination efficiently. After propagation of the urine-derived cells, iPSCs are generated by a modified transduction method of a commercially available Sendai virus vector system. FirstiPSC colonies may already be visible after 5 days, and can be picked after 10 days at the earliest. Routine clump passaging with enzyme-free dissociation buffer supports pluripotency of the generated iPSCs for more than 50 passages.

An mRNA-Based Multiple Antigenic Gene Expression System Delivered by Engineered Salmonella for Severe Fever with Thrombocytopenia Syndrome and Assessment of Its Immunogenicity and Protection Using a Human DC-SIGN-Transduced Mouse Model.

Currently, there are no commercial vaccines or therapeutics against severe fever with thrombocytopenia syndrome (SFTS) virus. This study explored an engineered Salmonella as a vaccine carrier to deliver a eukaryotic self-mRNA replicating vector, pJHL204. This vector expresses multiple SFTS virus antigenic genes for the nucleocapsid protein (NP), glycoprotein precursor (Gn/Gc), and nonstructural protein (NS) to induce host immune responses. The engineered constructs were designed and validated through 3D structure modeling. Western blot and qRT-PCR analyses of transformed HEK293T cells confirmed the delivery and expression of the vaccine antigens. Significantly, mice immunized with these constructs demonstrated a cell-mediated and humoral response as balanced Th1/Th2 immunity. The JOL2424 and JOL2425 delivering NP and Gn/Gc generated strong immunoglobulin IgG and IgM antibodies and high neutralizing titers. To further examine the immunogenicity and protection, we utilized a human DC-SIGN receptor transduced mouse model for SFTS virus infection by an adeno-associated viral vector system. Among the SFTSV antigen constructs, the construct with full-length NP and Gn/Gc and the construct with NP and selected Gn/Gc epitopes induced robust cellular and humoral immune responses. These were followed by adequate protection based on viral titer reduction and reduced histopathological lesions in the spleen and liver. In conclusion, these data indicate that recombinant attenuated Salmonella JOL2424 and JOL2425 delivering NP and Gn/Gc antigens of SFTSV are promising vaccine candidates that induce strong humoral and cellular immune responses and protection against SFTSV. Moreover, the data proved that the hDC-SIGN transduced mice as a worthy tool for immunogenicity study for SFTSV.

Microfluidic Chips: Emerging Technologies for Adoptive Cell Immunotherapy

Adoptive cell therapy (ACT) is a personalized therapy that has shown great success in treating hematologic malignancies in clinic, and has also demonstrated potential applications for solid tumors. The process of ACT involves multiple steps, including the separation of desired cells from patient tissues, cell engineering by virus vector systems, and infusion back into patients after strict tests to guarantee the quality and safety of the products. ACT is an innovative medicine in development; however, the multi-step method is time-consuming and costly, and the preparation of the targeted adoptive cells remains a challenge. Microfluidic chips are a novel platform with the advantages of manipulating fluid in micro/nano scales, and have been developed for various biological research applications as well as ACT. The use of microfluidics to isolate, screen, and incubate cells in vitro has the advantages of high throughput, low cell damage, and fast amplification rates, which can greatly simplify ACT preparation steps and reduce costs. Moreover, the customizable microfluidic chips fit the personalized demands of ACT. In this mini-review, we describe the advantages and applications of microfluidic chips for cell sorting, cell screening, and cell culture in ACT compared to other existing methods. Finally, we discuss the challenges and potential outcomes of future microfluidics-related work in ACT.

Abstract LB255: Benchmarking NGS integration site analysis methods in support of long-term safety monitoring of gene therapy products

Abstract The FDA Guidance to Industry on Long Term Follow-Up (LTFU) After Administration of Human Gene Therapy Products states the importance of longitudinal testing of gene products introduced into human subjects. Depending on the delivery mechanism, the therapeutic gene product may or may not integrate into the genome. Of particular interest are gene-product integrations near proto-oncogenes which might lead to malignancies. The FDA LTFU guidance states that recipients of an integrating gene therapy modality should be tracked for 15 years, while those receiving a non-integrating therapy modality should be tracked for 5 years. Therefore, advanced analytical methods are needed to identify, quantify, and track integration events across the genome. Here, we provide a comprehensive evaluation of methods leveraging next-generation sequencing approaches for genome-wide analysis of lentiviral integration events. Our analysis employed well-characterized standards consisting of varying copy number and known integration sites. ​ The approaches we characterized can be bucketed into two major groups: PCR amplification approaches and target capture-based approaches. All methods detected true positives with strong correlation to theoretical integration site dosage levels down to 1% allele frequency. Comparatively, PCR amplification-based approaches have lower data requirement per sample suggesting higher sensitivity, greater molecular capture, and lower limit of detection compared with target enrichment-based approaches. Target enrichment-based approaches can afford the flexibility to capture the integrated vector, which is of interest for characterizing partial integration events. While all methodologies performed well in our study, the choice of assay (or assays) for testing will depend on numerous factors including but not limited to the viral vector system and construct and starting material availability. Citation Format: Jeffrey M. Otto, Yongjun Fan, Guanhui Bao, David Corney, Ben Niu, Qiu Yu, Baursha Khan, Laure Turner, Chris Mozdzierz, Haythem Latif, Ginger Zhou. Benchmarking NGS integration site analysis methods in support of long-term safety monitoring of gene therapy products [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB255.

Nanotechnology-enabled gene delivery for cancer and other genetic diseases

Introduction Despite gene therapy is ideal for genetic abnormality-related diseases, the easy degradation, poor targeting, and inefficiency in entering targeted cells are plaguing the effective delivery of gene therapy. Viral and non-viral vectors have been used for delivering gene therapeutics in vivo by safeguarding nucleic acid agents to target cells and to reach the specific intracellular location. A variety of nanotechnology-enabled safe and efficient systems have been successfully developed to improve the targeting ability for effective therapeutic delivery of genetic drugs. Areas covered In this review, we outline the multiple biological barriers associated with gene delivery process, and highlight recent advances to gene therapy strategy in vivo, including gene correction, gene silencing, gene activation and genome editing. We point out current developments and challenges exist of non-viral and viral vector systems in association with chemical and physical gene delivery technologies and their potential for the future. Expert opinion This review focuses on the opportunities and challenges to various gene therapy strategy, with specific emphasis on overcoming the challenges through the development of biocompatibility and smart gene vectors for potential clinical application.

Adeno-associated virus vector system controlling capsid expression improves viral quantity and quality

Adeno-associated virus (AAV) vectors are promising tools for gene therapy. The current AAV vector system produces an abundance of empty capsids that are eliminated before clinical use, leading to increased costs for gene therapy. In the present study, we established an AAV production system that regulates the timing of capsid expression using a tetracycline-dependent promoter. Tetracycline-regulating capsid expression increased viral yield and reduced empty capsids in various serotypes without altering AAV vector infectivity invitro and invivo. The replicase expression pattern change observed in the developed AAV vector system improved viral quantity and quality, whereas timing control of capsid expression reduced empty capsids. These findings provide a new perspective on the development of AAV vector production systems in gene therapy.

Generation of integration-free Down syndrome and isogenic euploid human induced pluripotent stem cells

A pair of Down syndrome (DS) human iPSCs (hiPSCs) and isogenic euploid hiPSCs generated by using an integration-free Sendai viral vector system showed trisomy 21 (47; XY) and typical (46; XY) karyotype respectively. Pluripotency of both hiPSC lines was confirmed by pluripotency marker expression and three germ layer differentiation potentials.

Non-Covalent Linkage of Helper Functions to Dumbbell-Shaped DNA Vectors for Targeted Delivery.

Covalently closed dumbbell-shaped DNA delivery vectors comprising the double-stranded gene(s) of interest and single-stranded hairpin loops on both ends represent a safe, stable and efficacious alternative to viral and other non-viral DNA-based vector systems. As opposed to plasmids and DNA minicircles, dumbbells can be conjugated via the loops with helper functions for targeted delivery or imaging. Here, we investigated the non-covalent linkage of tri-antennary N-acetylgalactosamine (GalNAc3) or a homodimer of a CD137/4-1BB-binding aptamer (aptCD137-2) to extended dumbbell vector loops via complementary oligonucleotides for targeted delivery into hepatocytes or nasopharyngeal cancer cells. Enlarging the dumbbell loop size from 4 to 71 nucleotides for conjugation did not impair gene expression. GalNAc3 and aptCD137-2 residues were successfully attached to the extended dumbbell loop via complementary oligonucleotides. DNA and RNA oligonucleotide-based dumbbell-GalNAc3 conjugates were taken up from the cell culture medium by hepatoblastoma-derived human tissue culture cells (HepG2) with comparable efficiency. RNA oligonucleotide-linked conjugates triggered slightly higher levels of gene expression, presumably due to the RNaseH-mediated linker cleavage, the release of the dumbbell from the GalNAc3 residue and more efficient nuclear targeting of the unconjugated dumbbell DNA. The RNaseH-triggered RNA linker cleavage was confirmed in vitro. Finally, we featured dumbbell vectors expressing liver cancer cell-specific RNA trans-splicing-based suicide RNAs with GalNAc3 residues. Dumbbells conjugated with two GalNAc3 residues triggered significant levels of cell death when added to the cell culture medium. Dumbbell vector conjugates can be explored for targeted delivery and gene therapeutic applications.

Regulatory Trends in the Nonclinical Development of Viral Vector-Based Gene Therapies: A Benchmark Analysis of Approved Products

As it is often the case with innovative technologies, regulatory agencies are highly demanding in product safety demonstration from those pioneering breakthrough therapy products. Since the first historically approved gene therapy medicinal product (Gencidine in 2003) by the Chinese National Medicinal Products Administration, gene therapy medicinal products have slowly been emerging in other regions, as illustrated by the first European-approved gene therapy medicinal product (Glybera in 2012) and the first US-approved product (Imlygic in 2015). From then, with the rise of new molecular technologies (e.g., non-viral and viral vector systems), an exponential growth of gene therapies development could be gauged with, for example, the approval of more than thirty gene therapies between 2016-2022. Using a method based on Preferred Reporting Items for Systematic reviews and Meta-Analyses principle, throughout different literature databases, this review is restricted to the evaluation of viral vector-based gene therapy medicinal products (VV-GTMPs). It also considered relevant guidelines and public assessment reports issued by the EMA and / or the FDA on the products these agencies approved. Then, a benchmark was performed to help stakeholders to identify regulatory trends and to design appropriate nonclinical programs establishing the benefit / risk ratio for patients to be enrolled in clinical trials. The analysis was focused on the nonclinical activities (pharmacology, biodistribution / persistence / shedding, and toxicology) performed by Applicants / Sponsors. As of 30 March 2023, 18 VV-GTMPs have been authorized by the EMA and 14 by the FDA to treat either orphan diseases or limited number of oncology patients. The majority of these therapies are based on adeno-associated or retroviral viruses (often lentiviruses able to transfect hematopoietic CD34+ cells or T-cells). Based on an analysis of the ongoing clinical trials, there is now a trend for developing gene therapies for larger patient populations. In conclusion, given the VV-GTMPs diversity and targeted indications, a “one-size fits all” nonclinical development plan cannot be considered by default. Instead, individual, risk-based, tailored nonclinical development programs appear more appropriate to assess such products, taking into consideration the lessons learned from the past. In such fast-evolving environment, regulatory agencies need to adapt their evaluation process very rapidly.

In vivo editing of the pan-endothelium by immunity evading simian adenoviral vector

Biological applications deriving from the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 site-specific nuclease system continue to impact and accelerate gene therapy strategies. Safe and effective in vivo co-delivery of the CRISPR/Cas9 system to target somatic cells is essential in the clinical therapeutic context. Both non-viral and viral vector systems have been applied for this delivery matter. Despite elegant proof-of-principle studies, available vector technologies still face challenges that restrict the application of CRISPR/Cas9-facilitated gene therapy. Of note, the mandated co-delivery of the gene-editing components must be accomplished in the potential presence of pre-formed anti-vector immunity. Additionally, methods must be sought to limit the potential of off-target editing. To this end, we have exploited the molecular promiscuities of adenovirus (Ad) to address the key requirements of CRISPR/Cas9-facilitated gene therapy. In this regard, we have endeavored capsid engineering of a simian (chimpanzee) adenovirus isolate 36 (SAd36) to achieve targeted modifications of vector tropism. The SAd36 vector with the myeloid cell-binding peptide (MBP) incorporated in the capsid has allowed selective in vivo modifications of the vascular endothelium. Importantly, vascular endothelium can serve as an effective non-hepatic cellular source of deficient serum factors relevant to several inherited genetic disorders. In addition to allowing for re-directed tropism, capsid engineering of nonhuman primate Ads provide the means to circumvent pre-formed vector immunity. Herein we have generated a SAd36. MBP vector that can serve as a single intravenously administered agent allowing effective and selective in vivo editing for endothelial target cells of the mouse spleen, brain and kidney. Data availabilityThe data that support the findings of this study are available from the corresponding author upon reasonable request.

CRISPR-Cas9 gene editing and human diseases.

CRISPR/Cas-9 mediated genome editing has recently emerged as a potential and innovative technology in therapeutic development and biomedical research. Several recent studies have been performed to understand gene modification techniques in obtaining effective ex vivo results. Generally, the disease targets for gene correction will be in specific organs, so understanding the complete potential of genomic treatment methods is essential. From such a perspective, the present review revealed the significant importance of the CRISPR/ Cas9 delivery system. Both the promising gene-editing delivery systems, such as synthetic (non-viral) and viral vector systems are discussed in this review. In addition, this paper attempted to summarize the tissue-specific and organ-specific mRNA delivery systems that provide possible research information for future researchers. Further, the major challenges of the CRISPR/Cas9 system, such as off-target delivery, immunogenicity, and limited packaging, were also elucidated. Accordingly, this review illustrated a wide range of clinical applications associated with the efficient delivery of CRISPR/ Cas9 gene-editing. Moreover, this article emphasizes the role of the CRISPR/Cas9 system in treating Intra Cerebral haemorrhage (ICH), thereby suggesting future researchers to adopt more clinical trials on this breakthrough delivery system.