PCR-Based Analytical Methods for Quantification and Quality Control of Recombinant Adeno-Associated Viral Vector Preparations

Recombinant adeno-associated viral vectors (rAAV) represent a gene therapy tool of ever-increasing importance. Their utilization as a delivery vehicle for gene replacement, silencing and editing, among other purposes, demonstrate considerable versatility. Emerging vector utilization in various experimental, preclinical and clinical applications establishes the necessity of producing and characterizing a wide variety of rAAV preparations. Critically important characteristics concerning quality control are rAAV titer quantification and the detection of impurities. Differences in rAAV constructs necessitate the development of highly standardized quantification assays to make direct comparisons of different preparations in terms of assembly or purification efficiency, as well as experimental or therapeutic dosages. The development of universal methods for impurities quantification is rather complicated, since variable production platforms are utilized for rAAV assembly. However, general agreements also should be achieved to address this issue. The majority of methods for rAAV quantification and quality control are based on PCR techniques. Despite the progress made, increasing evidence concerning high variability in titration assays indicates poor standardization of the methods undertaken to date. This review summarizes successes in the field of rAAV quality control and emphasizes ongoing challenges in PCR applications for rAAV characterization. General considerations regarding possible solutions are also provided.

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CRISPR/Cas9 Delivery Potentials in Alzheimer’s Disease Management: A Mini Review

Pharmaceutics ◽

10.3390/pharmaceutics12090801 ◽

2020 ◽

Vol 12 (9) ◽

pp. 801

Author(s):

Amira Sayed Hanafy ◽

Susanne Schoch ◽

Alf Lamprecht

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Viral Vectors ◽

Gene Editing ◽

Gene Sequence ◽

Viral Vector ◽

Delivery Vehicle ◽

Effective Delivery ◽

In Vivo Delivery

Alzheimer’s disease (AD) is the most common dementia disorder. While genetic mutations account for only 1% of AD cases, sporadic AD resulting from a combination of genetic and risk factors constitutes >90% of the cases. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein (Cas9) is an impactful gene editing tool which identifies a targeted gene sequence, creating a double-stranded break followed by gene inactivation or correction. Although CRISPR/Cas9 can be utilized to irreversibly inactivate or correct faulty genes in AD, a safe and effective delivery system stands as a challenge against the translation of CRISPR therapeutics from bench to bedside. While viral vectors are efficient in CRISPR/Cas9 delivery, they might introduce fatal side effects and immune responses. As non-viral vectors offer a better safety profile, cost-effectiveness and versatility, they can be promising for the in vivo delivery of CRISPR/Cas9 therapeutics. In this minireview, we present an overview of viral and non-viral vector based CRISPR/Cas9 therapeutic strategies that are being evaluated on pre-clinical AD models. Other promising non-viral vectors that can be used for genome editing in AD, such as nanoparticles, nanoclews and microvesicles, are also discussed. Finally, we list the formulation and technical aspects that must be considered in order to develop a successful non-viral CRISPR/Cas9 delivery vehicle.

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VipariNama: RNA viral vectors to rapidly elucidate the relationship between gene expression and phenotype

Plant Physiology ◽

10.1093/plphys/kiab197 ◽

2021 ◽

Author(s):

Arjun Khakhar ◽

Cecily Wang ◽

Ryan Swanson ◽

Sydney Stokke ◽

Furva Rizvi ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Viral Vectors ◽

Viral Vector ◽

Tobacco Rattle Virus ◽

Plant Size ◽

Great Promise ◽

Attractive Alternative ◽

Gibberellin Biosynthesis ◽

In Planta

Abstract Synthetic transcription factors have great promise as tools to help elucidate relationships between gene expression and phenotype by allowing tunable alterations of gene expression without genomic alterations of the loci being studied. However, the years-long timescales, high cost, and technical skill associated with plant transformation have limited their use. In this work we developed a technology called VipariNama (ViN) in which vectors based on the Tobacco Rattle Virus (TRV) are used to rapidly deploy Cas9-based synthetic transcription factors and reprogram gene expression in planta. We demonstrate that ViN vectors can implement activation or repression of multiple genes systemically and persistently over several weeks in Nicotiana benthamiana, Arabidopsis (Arabidopsis thaliana), and tomato (Solanum lycopersicum). By exploring strategies including RNA scaffolding, viral vector ensembles, and viral engineering, we describe how the flexibility and efficacy of regulation can be improved. We also show how this transcriptional reprogramming can create predictable changes to metabolic phenotypes, such as gibberellin biosynthesis in N. benthamiana and anthocyanin accumulation in Arabidopsis, as well as developmental phenotypes, such as plant size in N. benthamiana, Arabidopsis, and tomato. These results demonstrate how ViN vector-based reprogramming of different aspects of gibberellin signaling can be used to engineer plant size in a range of plant species in a matter of weeks. In summary, VipariNama accelerates the timeline for generating phenotypes from over a year to just a few weeks, providing an attractive alternative to transgenesis for synthetic transcription factor-enabled hypothesis testing and crop engineering.

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How Far Are Non-Viral Vectors to Come of Age and Reach Clinical Translation in Gene Therapy?

International Journal of Molecular Sciences ◽

10.3390/ijms22147545 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7545

Author(s):

Myriam Sainz-Ramos ◽

Idoia Gallego ◽

Ilia Villate-Beitia ◽

Jon Zarate ◽

Iván Maldonado ◽

...

Keyword(s):

Gene Therapy ◽

Clinical Practice ◽

Gene Delivery ◽

Viral Vectors ◽

Viral Vector ◽

Clinical Success ◽

Genetic Material ◽

Viral Gene ◽

Promising Alternative ◽

Vector Systems

Efficient delivery of genetic material into cells is a critical process to translate gene therapy into clinical practice. In this sense, the increased knowledge acquired during past years in the molecular biology and nanotechnology fields has contributed to the development of different kinds of non-viral vector systems as a promising alternative to virus-based gene delivery counterparts. Consequently, the development of non-viral vectors has gained attention, and nowadays, gene delivery mediated by these systems is considered as the cornerstone of modern gene therapy due to relevant advantages such as low toxicity, poor immunogenicity and high packing capacity. However, despite these relevant advantages, non-viral vectors have been poorly translated into clinical success. This review addresses some critical issues that need to be considered for clinical practice application of non-viral vectors in mainstream medicine, such as efficiency, biocompatibility, long-lasting effect, route of administration, design of experimental condition or commercialization process. In addition, potential strategies for overcoming main hurdles are also addressed. Overall, this review aims to raise awareness among the scientific community and help researchers gain knowledge in the design of safe and efficient non-viral gene delivery systems for clinical applications to progress in the gene therapy field.

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Clinical applications and quality control for the clatterbridge cyclotron trials

Clinical Oncology ◽

10.1016/s0936-6555(89)80012-3 ◽

1989 ◽

Vol 1 (1) ◽

pp. 49

Keyword(s):

Quality Control ◽

Clinical Applications

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Abstract 603: Effective And Sustained ACE2 Delivery Using Minicircles Technology

Hypertension ◽

10.1161/hyp.62.suppl_1.a603 ◽

2013 ◽

Vol 62 (suppl_1) ◽

Author(s):

Jan Wysocki ◽

Philipp K Haber ◽

Minghao Ye ◽

Christoph Maier ◽

Mark J Osborn ◽

...

Keyword(s):

Viral Vectors ◽

Gene Replacement ◽

Ang Ii ◽

Hydrodynamic Approach ◽

New Gene ◽

Cloned Cdna ◽

Dose Dependent ◽

Delivery Technology

Chronic and sustained amplification of ACE2 activity in vivo has required the development of transgenic mice or the use of viral vectors. Minicircle is a new gene delivery technology which is resistant to gene silencing, and therefore represents an attractive platform for gene replacement strategies in vivo . Here we cloned cDNA of soluble mouse ACE2 into a circular expression cassette and the resulting ACE2 minicircle (MC) was injected to female FVB mice using iv. hydrodynamic approach (10ug or 30ug/mouse). At 3-7d after MC administration, serum ACE2 activity in mice that received 10ug ACE2MC (n=9) was over 100-fold higher than in controls (n=9) (138±48 vs 0.7±0.2 RFU/uL/hr) and in ACE2MC mice (30ug) (n=8) was almost 1000-fold higher than in controls (n=14) (480 ±153 vs 0.5±0.1 RFU/uL/hr, respectively). Mice that received 10 ug ACE2MC were followed for consecutive serum ACE2 activity monitoring, BP measurements and plasma Ang levels. The increase in serum ACE2 activity was sustained until the end of the study (up to 82 days) (Figure). Despite such a marked increase in serum ACE2 activity in ACE2MC mice, conscious SBP was not different from controls (137±8 vs 138±7 mmHg, respectively). At the end of the study, when Ang II was infused acutely (0.2 ug/kg BW i.p.), the increase in plasma Ang II in ACE2MC mice was significantly reduced compared to control mice (915±154 vs 1420±131 fmoL/mL, p<0.05). Mini-circle delivery of ACE2 results in a dose-dependent and sustained long-term increase in serum ACE2 that efficiently degrades plasma Ang II. Extremely high increases in serum ACE2 activity do not reduce BP probably due to activation of non-ACE2 dependent compensatory Ang-hydrolyzing pathways.

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Recent Advances in Clinical Trials of HCV Vaccines

Journal of Applied Virology ◽

10.21092/jav.v3i1.48 ◽

2014 ◽

Vol 3 (1) ◽

pp. 10 ◽

Cited By ~ 2

Author(s):

Yongneng Luo ◽

Limin Jiang ◽

Zi'an Mao

Keyword(s):

Clinical Trials ◽

Viral Vectors ◽

Vaccine Development ◽

Viral Vector ◽

Core Protein ◽

Therapeutic Vaccine ◽

Hcv Infection ◽

Effective Vaccine ◽

Protein Subunit ◽

Preclinical Development

<p> Hepatitis C virus infects nearly 3% of the global population, and spreads to 3-4 million new people annually. HCV infection is a leading cause of liver cirrhosis, hepatocellular carcinoma, and end-stage liver diseases and causes liver-related death in more than 300,000 people each year. Unfortunately, there is currently no vaccine for HCV prevention (prophylactic vaccine) or treatment (therapeutic vaccine). Circulating HCV is genetically diverse, and therefore a broadly effective vaccine must target conserved T- and B-cell epitopes of the virus and induce strong cross-reactive CD4+/CD8+ T-cell and neutralizing antibody responses in preventing or clearing HCV infection. So far, a few of vaccine development approaches are successful and some of the HCV vaccine candidates have reached human clinical trials, including those modalities mainly based on recombinant proteins (envelope proteins and core protein subunit), synthetic peptides, DNA (plasmid) and viral vectors (virosome). Encouraging results were obtained for those HCV vaccine formulations consisting of prime-boost regimen involving a live recombinant viral vector vaccine alone or in combination with DNA or subunit vaccine. Among several other vaccine strategies under preclinical development, the most promising one is virus like particle based vaccine that will be moving into human studies soon.</p>

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Quality Control of Cellular Therapy Products and Viral Vectors

10.1007/978-3-030-75537-9_12 ◽

2021 ◽

pp. 209-223

Author(s):

Adrian P. Gee

Keyword(s):

Quality Control ◽

Viral Vectors ◽

Cellular Therapy

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Hatchery Vaccination Quality Control of Herpesvirus of Turkey-Infectious Bursal Disease HVT-IBD Viral Vector Vaccine Application by Specific qPCR

International Journal of Poultry Science ◽

10.3923/ijps.2012.570.576 ◽

2012 ◽

Vol 11 (9) ◽

pp. 570-576 ◽

Cited By ~ 1

Author(s):

Stephane Lemiere ◽

Francisco Perozo ◽

Blandine de Saint-Vis ◽

Jennifer Diasparra ◽

Arnaud Carlotti ◽

...

Keyword(s):

Quality Control ◽

Viral Vector ◽

Infectious Bursal Disease ◽

Vector Vaccine ◽

Bursal Disease ◽

Viral Vector Vaccine

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Layered Double Hydroxide as a Potent Non-viral Vector for Nucleic Acid Delivery Using Gene-Activated Scaffolds for Tissue Regeneration Applications

Pharmaceutics ◽

10.3390/pharmaceutics12121219 ◽

2020 ◽

Vol 12 (12) ◽

pp. 1219

Author(s):

Lara S. Costard ◽

Domhnall C. Kelly ◽

Rachael N. Power ◽

Christopher Hobbs ◽

Sonia Jaskaniec ◽

...

Keyword(s):

Gene Therapy ◽

Layered Double Hydroxide ◽

Viral Vectors ◽

Viral Vector ◽

Nucleic Acid Delivery ◽

Safe Alternative ◽

Transfection Efficacy ◽

Mass Ratios ◽

Low Cytotoxicity ◽

Double Hydroxide

Nonviral vectors offer a safe alternative to viral vectors for gene therapy applications, albeit typically exhibiting lower transfection efficiencies. As a result, there remains a significant need for the development of a nonviral delivery system with low cytotoxicity and high transfection efficacy as a tool for safe and transient gene delivery. This study assesses MgAl-NO3 layered double hydroxide (LDH) as a nonviral vector to deliver nucleic acids (pDNA, miRNA and siRNA) to mesenchymal stromal cells (MSCs) in 2D culture and using a 3D tissue engineering scaffold approach. Nanoparticles were formulated by complexing LDH with pDNA, microRNA (miRNA) mimics and inhibitors, and siRNA at varying mass ratios of LDH:nucleic acid. In 2D monolayer, pDNA delivery demonstrated significant cytotoxicity issues, and low cellular transfection was deemed to be a result of the poor physicochemical properties of the LDH–pDNA nanoparticles. However, the lower mass ratios required to successfully complex with miRNA and siRNA cargo allowed for efficient delivery to MSCs. Furthermore, incorporation of LDH–miRNA nanoparticles into collagen-nanohydroxyapatite scaffolds resulted in successful overexpression of miRNA in MSCs, demonstrating the development of an efficacious miRNA delivery platform for gene therapy applications in regenerative medicine.

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