scholarly journals Improved Genome Packaging Efficiency of AAV Vectors Using Rep Hybrids

2021 ◽  
Author(s):  
Mario Mietzsch ◽  
Courtnee Eddington ◽  
Ariana Jose ◽  
Jane Hsi ◽  
Paul Chipman ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Production Systems ◽  
The Other ◽  
Genome Packaging ◽  
Reading Frame ◽  
Gene Therapies ◽  
Vector Genome ◽  
Packaging Efficiency ◽  
Raav Production

Recombinant Adeno-associated viruses (rAAVs) are one of the most commonly used vectors for a variety of gene therapy applications. In the last two decades research focused primarily on the characterization and isolation of new cap genes resulting in hundreds of natural and engineered AAV capsid variants while the rep gene, the other major AAV open reading frame, has been less studied. This is due to the fact that the rep gene from AAV serotype 2 (AAV2) enables the ssDNA packaging of recombinant genomes into most AAV serotype and engineered capsids. However, a major byproduct of all vector productions is empty AAV capsids, lacking the encapsidated vector genome, especially for non-AAV2 vectors. Despite the packaging process being considered the rate-limiting step for rAAV production, none of the rep genes from the other AAV serotypes have been characterized for their packaging efficiency. Thus, in this study AAV2 rep was replaced with the rep gene of a select number of AAV serotypes. However, this led to a lowering of capsid protein expression, relative to the standard AAV2- rep system. In further experiments the 3’end of the AAV2 rep gene was reintroduced to promote increased capsid expression and a series of chimeras between the different AAV Rep proteins were generated and characterized for their vector genome packaging ability. The utilization of these novel Rep hybrids increased the percentage of genome containing (full) capsids ∼2-4-fold for all of the non-AAV2 serotypes tested. Thus, these Rep chimeras could revolutionize rAAV production. Importance A major byproduct of all Adeno-associated virus (AAV) vector production systems are “empty” capsids, void of the desired therapeutic gene, and thus do not provide any curative benefit for the treatment of the targeted disease. In fact, empty capsids can potentially elicit additional immune responses in vivo gene therapies if not removed by additional purification steps. Thus, there is a need to increase the genome packaging efficiency and reduce the number of empty capsids from AAV biologics. The novel Rep hybrids from different AAV serotypes described in this study are capable of reducing the percentage of empty capsids in all tested AAV serotypes and improve overall yields of genome-containing AAV capsids at the same time. They can likely be integrated easily into existing AAV manufacturing protocols to optimize the production of the generated AAV gene therapy products.

scholarly journals Improved Genome Packaging Efficiency of AAV Vectors Using Rep Hybrids

2021 ◽  
Author(s):  
Mario Mietzsch ◽  
Courtnee Eddington ◽  
Ariana Jose ◽  
Jane Hsi ◽  
Paul Chipman ◽  
...  
Keyword(s):  
The Other ◽  
Genome Packaging ◽  
Reading Frame ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Vector Genome ◽  
Rep Proteins ◽  
Packaging Efficiency ◽  
Raav Production ◽  
Rate Limiting

Recombinant Adeno-associated viruses (rAAVs) are one of the most commonly used vectors for a variety of gene therapy applications. In the last two decades research focused primarily on the characterization and isolation of new cap genes resulting in hundreds of natural and engineered AAV capsid variants while the rep gene, the other major AAV open reading frame, has been less studied. This is due to the fact that the rep gene from AAV serotype 2 (AAV2) enables the ssDNA packaging of recombinant genomes into most AAV serotype and engineered capsids. However, a major byproduct of all vector productions is empty AAV capsids, lacking the encapsidated vector genome, especially for non-AAV2 vectors. Despite the packaging process being considered the rate-limiting step for rAAV production, none of the rep genes from the other AAV serotypes have been characterized for their packaging efficiency. Thus, in this study AAV2 rep was replaced with the rep gene of a select number of AAV serotypes. However, this led to a lowering of capsid protein expression, relative to the standard AAV2-rep system. In further experiments the 3’end of the AAV2 rep gene was reintroduced to promote increased capsid expression and a series of chimeras between the different AAV Rep proteins were generated and characterized for their vector genome packaging ability. The utilization of these novel Rep hybrids increased the percentage of genome containing (full) capsids ~2-4-fold for all of the non-AAV2 serotypes tested. Thus, these Rep chimeras could revolutionize rAAV production.

Gene therapy comes of age

Science ◽  
2018 ◽  
Vol 359 (6372) ◽  
pp. eaan4672 ◽  
Author(s):  
Cynthia E. Dunbar ◽  
Katherine A. High ◽  
J. Keith Joung ◽  
Donald B. Kohn ◽  
Keiya Ozawa ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
The United States ◽  
Genetically Engineered ◽  
Drug Status ◽  
Hematopoietic Stem ◽  
Gene Therapies ◽  
Engineered T Cells ◽  
Near Future

After almost 30 years of promise tempered by setbacks, gene therapies are rapidly becoming a critical component of the therapeutic armamentarium for a variety of inherited and acquired human diseases. Gene therapies for inherited immune disorders, hemophilia, eye and neurodegenerative disorders, and lymphoid cancers recently progressed to approved drug status in the United States and Europe, or are anticipated to receive approval in the near future. In this Review, we discuss milestones in the development of gene therapies, focusing on direct in vivo administration of viral vectors and adoptive transfer of genetically engineered T cells or hematopoietic stem cells. We also discuss emerging genome editing technologies that should further advance the scope and efficacy of gene therapy approaches.

scholarly journals Generation of an Adenovirus Vector Lacking E1, E2a, E3, and All of E4 except Open Reading Frame 3

1999 ◽  
Vol 73 (7) ◽  
pp. 6048-6055 ◽  
Author(s):  
Mario I. Gorziglia ◽  
Claudia Lapcevich ◽  
Soumitra Roy ◽  
Qiang Kang ◽  
Mike Kadan ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Liver Toxicity ◽  
Adenovirus Vector ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Adenovirus Vectors ◽  
Virus Promoter

ABSTRACT Toxicity and immunity associated with adenovirus backbone gene expression is an important hurdle to overcome for successful gene therapy. Recent efforts to improve adenovirus vectors for in vivo use have focused on the sequential deletion of essential early genes. Adenovirus vectors have been constructed with the E1 gene deleted and with this deletion in combination with an E2a, E2b, or E4 deletion. We report here a novel vector (Av4orf3nBg) lacking E1, E2a, and all of E4 except open reading frame 3 (ORF3) and expressing a β-galactosidase reporter gene. This vector was generated by transfection of a plasmid carrying the full-length vector sequence into A30.S8 cells that express E1 and E2a but not E4. Production was subsequently performed in an E1-, E2a-, and E4-complementing cell line. We demonstrated with C57BL/6 mice that the Av4orf3nBg vector effected gene transfer with an efficiency comparable to that of the Av3nBg (wild-type E4) vector but that the former exhibited a higher level of β-galactosidase expression. This observation suggests that E4 ORF3 alone is able to enhance RNA levels from the β-galactosidase gene when the Rous sarcoma virus promoter is used to drive transgene expression in the mouse liver. In addition, we observed less liver toxicity in mice injected with the Av4orf3nBg vector than those injected with the Av3nBg vector at a comparable DNA copy number per cell. This study suggests that the additional deletion of E4 in an E1 and E2a deletion background may be beneficial in decreasing immunogenicity and improving safety and toxicity profiles, as well as increasing transgene capacity and expression for liver-directed gene therapy.

scholarly journals Modeling human lymphoid precursor cell gene therapy in the SCID-hu mouse

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1393-1398 ◽  
Author(s):  
RK Akkina ◽  
JD Rosenblatt ◽  
AG Campbell ◽  
IS Chen ◽  
JA Zack
Keyword(s):  
Gene Therapy ◽  
In Vivo Model ◽  
Hematopoietic Stem ◽  
Gene Therapies ◽  
Human Cd34 ◽  
Human T Lymphocyte ◽  
Thymocyte Differentiation ◽  
Immunodeficiency Syndrome

Abstract Gene therapy of human T-lymphocyte disorders, including acquired immunodeficiency syndrome (AIDS), would be greatly facilitated by the development of an in vivo system in which transduced human hematopoietic stem cells can be used to reconstitute the T-lymphoid compartment. Here we use the SCID-hu mouse as a recipient for human CD34+ hematopoietic progenitor cells transduced in vitro with a retroviral vector carrying the neomycin resistance gene (neoR). The transduced cells engraft and reconstitute the lymphoid compartments of the human thymus implant with as few as 5 x 10(4) CD34+ cells. The neoR gene was expressed at low levels in human thymocytes and there was no apparent effect on thymocyte differentiation as a result of vector transduction. Thus, this SCID-hu mouse system is the first in vivo model showing human thymopoiesis after transduction of exogenous vectors, and should allow preclinical testing of gene therapeutic reagents designed to function in human cells of the T-lymphoid lineage. Because human immunodeficiency virus type 1 infection induces depletion of human thymocytes in SCID-hu mice, this system may be particularly valuable in evaluating efficacy of gene therapies to combat AIDS.

The Effectiveness of Nanoparticles on Gene Therapy for Glioblastoma Cells Apoptosis: A Systematic Review

2021 ◽  
Vol 21 ◽  
Author(s):  
Firoozeh Alavian ◽  
Sorayya Ghasemi
Keyword(s):  
Systematic Review ◽  
Gene Therapy ◽  
Data Extraction ◽  
Gene Therapies ◽  
Gene Therapy Approach ◽  
Therapy Studies ◽  
Present Systematic Review

Background: Glioblastoma multiforme (GBM) is the most common and fatal type of glioma. Nanoparticles (NPs) are using new approaches for the delivery of gene therapy in the treatment of GBM. Introduction: This article was designed to review the efficacy of NPs as the targeted carriers in the gene therapy aimed at apoptosis in GBM. Method: The appropriate keywords such as nanoparticle, glioblastoma, gene therapy, apoptosis, and the related words were used to search from PubMed, ISI Web of Science, and Scopus for relevant publications up to September 4, 2020, with no language restrictions. The present systematic review was performed based on PRISMA protocol and reviewed the articles evaluating the effects of nanoparticles, carriers of various gene therapies essentials, on GBM cells apoptosis in vitro and in vivo. The selected articles were considered using specific scores on the quality of the articles. Data extraction and quality valuation were performed by two reviewers. Result: Of 101 articles retrieved, forty-two met the inclusion criteria and were, therefore, subjected to the final deduction. The most widely used NP in GBM gene therapy studies is polyamidoamine (PAMAM). The most common gene therapy approach for apoptosis in GBM is using siRNAs. Conclusions: In conclusion, these studies validated that NPs could be a practical choices to enhance the efficiency and specific delivery in gene therapies for GBM cell apoptosis. However, the choice of NP type and gene therapy mechanism affect the GBM cell apoptotic efficiency.

scholarly journals The Structure of an AAV5-AAVR Complex at 2.5 Å Resolution: Implications for Cellular Entry and Immune Neutralization of AAV Gene Therapy Vectors

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1326
Author(s):  
Mark A. Silveria ◽  
Edward E. Large ◽  
Grant M. Zane ◽  
Tommi A. White ◽  
Michael S. Chapman
Keyword(s):  
Gene Therapy ◽  
Binding Sites ◽  
Polycystic Kidney ◽  
Adeno Associated Virus ◽  
Gene Therapies ◽  
The Us ◽  
Gene Therapy Vectors ◽  
Cellular Entry ◽  
Resolution Structure

Adeno-Associated Virus is the leading vector for gene therapy. Although it is the vector for all in vivo gene therapies approved for clinical use by the US Food and Drug Administration, its biology is still not yet fully understood. It has been shown that different serotypes of AAV bind to their cellular receptor, AAVR, in different ways. Previously we have reported a 2.4Å structure of AAV2 bound to AAVR that shows ordered structure for only one of the two AAVR domains with which AAV2 interacts. In this study we present a 2.5Å resolution structure of AAV5 bound to AAVR. AAV5 binds to the first polycystic kidney disease (PKD) domain of AAVR that was not ordered in the AAV2 structure. Interactions of AAV5 with AAVR are analyzed in detail, and the implications for AAV2 binding are explored through molecular modeling. Moreover, we find that binding sites for the antibodies ADK5a, ADK5b, and 3C5 on AAV5 overlap with the binding site of AAVR. These insights provide a structural foundation for development of gene therapy agents to better evade immune neutralization without disrupting cellular entry.

scholarly journals Production and Characterization of Improved Adenovirus Vectors with the E1, E2b, and E3 Genes Deleted

1998 ◽  
Vol 72 (2) ◽  
pp. 926-933 ◽  
Author(s):  
Andrea Amalfitano ◽  
Michael A. Hauser ◽  
Huimin Hu ◽  
Delila Serra ◽  
Catherine R. Begy ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Late Gene Expression ◽  
Quiescent Cells ◽  
293 Cells ◽  
Vector Genome ◽  
Gene Functions ◽  
Viral Genes

ABSTRACT Adenovirus (Ad)-based vectors have great potential for use in the gene therapy of multiple diseases, both genetic and nongenetic. While capable of transducing both dividing and quiescent cells efficiently, Ad vectors have been limited by a number of problems. Most Ad vectors are engineered such that a transgene replaces the Ad E1a, E1b, and E3 genes; subsequently the replication-defective vector can be propagated only in human 293 cells that supply the deleted E1 gene functions intrans. Unfortunately, the use of high titers of E1-deleted vectors has been repeatedly demonstrated to result in low-level expression of viral genes still resident in the vector. In addition, the generation of replication-competent Ad (RCA) by recombination events with the E1 sequences residing in 293 cells further limits the usefulness of E1-deleted Ad vectors. We addressed these problems by isolating new Ad vectors deleted for the E1, E3, and the E2b gene functions. The new vectors can be readily grown to high titers and have several improvements, including an increased carrying capacity and a theoretically decreased risk for generating RCA. We have also demonstrated that the further block to Ad vector replication afforded by the deletion of both the E1 and E2b genes significantly diminished Ad late gene expression in comparison to a conventional E1-deleted vector, without destabilization of the modified vector genome. The results suggested that these modified vectors may be very useful both for in vitro and in vivo gene therapy applications.

scholarly journals Adenovirus Vectors with the 100K Gene Deleted and Their Potential for Multiple Gene Therapy Applications

2001 ◽  
Vol 75 (13) ◽  
pp. 5913-5920 ◽  
Author(s):  
B. L. Hodges ◽  
H. K. Evans ◽  
R. S. Everett ◽  
E. Y. Ding ◽  
D. Serra ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Copy Number ◽  
Late Phase ◽  
Genome Replication ◽  
Successful Completion ◽  
Late Gene Expression ◽  
Multiple Gene ◽  
Late Genes ◽  
Vector Genome

ABSTRACT The 100K protein has a number of critical roles vital for successful completion of the late phases of the adenovirus (Ad) life cycle. We hypothesized that the introduction of deletions within the 100K gene would allow for the production of a series of new classes of Ad vector, including one that is replication competent but blocked in the ability to carry out many late-phase Ad functions. Such a vector would have potential for several gene therapy applications, based upon its ability to increase the copy number of the transgene encoded by the vector (via genome replication) while decreasing the side effects associated with Ad late gene expression. To efficiently produce 100K-deleted Ad ([100K−]Ad) vectors, an E1- and 100K-complementing cell line (K-16) was successfully isolated. Transfection of an [E1−,100K−]Ad vector genome into the K-16 cells readily yielded high titers of the vector. After infection of noncomplementing cells, we demonstrated that [100K−]Ad vectors have a significantly decreased ability to express several Ad late genes. Additionally, if the E1 gene was present in the infected noncomplementing cells, [100K−]Ad vectors were capable of replicating their genomes to high copy number, but were significantly blocked in their ability to efficiently encapsidate the replicated genomes. Injection of an [E1−,100K−]Ad vector in vivo also correlated with significantly decreased hepatotoxicity, as well as prolonged vector persistence. In summary, the unique properties of [100K−]Ad vectors suggest that they may have utility in a variety of gene therapy applications.

scholarly journals Cas9-directed immune tolerance in humans—a model to evaluate regulatory T cells in gene therapy?

Gene Therapy ◽  
2021 ◽  
Author(s):  
Dimitrios Laurin Wagner ◽  
Lena Peter ◽  
Michael Schmueck-Henneresse
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
Regulatory T Cells ◽  
Immune Tolerance ◽  
Neutralizing Antibodies ◽  
Treg Cells ◽  
Gene Therapies ◽  
Early Results ◽  
Cell Immunity

AbstractThe dichotomic nature of the adaptive immune response governs the outcome of clinical gene therapy. On the one hand, neutralizing antibodies and cytotoxic T cells can have a dramatic impact on the efficacy and safety of human gene therapies. On the other hand, regulatory T cells (Treg) can promote tolerance toward transgenes thereby enabling long-term benefits of in vivo gene therapy after a single administration. Pre-existing antibodies and T cell immunity has been a major obstacle for in vivo gene therapies with viral vectors. As CRISPR-Cas9 gene editing advances toward the clinics, the technology’s inherent immunogenicity must be addressed in order to guide clinical treatment decisions. This review summarizes the recent evidence on Cas9-specific immunity in humans—including early results from clinical trials—and discusses the risks for in vivo gene therapies. Finally, we focus on solutions and highlight the potential role of Cas9-specific Treg cells to promote immune tolerance. As a “beneficial alliance” beyond Cas9-immunity, antigen-specific Treg cells may serve as a living and targeted immunosuppressant to increase safety and efficacy of gene therapy.

scholarly journals Evolving AAV-delivered therapeutics towards ultimate cures

2021 ◽  
Author(s):  
Xiangjun He ◽  
Brian Anugerah Urip ◽  
Zhenjie Zhang ◽  
Chun Christopher Ngan ◽  
Bo Feng
Keyword(s):  
Gene Therapy ◽  
Gene Editing ◽  
Treatment Options ◽  
Genetic Diseases ◽  
The United States ◽  
European Medicines Agency ◽  
Gene Therapies ◽  
Therapeutic Modalities ◽  
United States Food

AbstractGene therapy has entered a new era after decades-long efforts, where the recombinant adeno-associated virus (AAV) has stood out as the most potent vector for in vivo gene transfer and demonstrated excellent efficacy and safety profiles in numerous preclinical and clinical studies. Since the first AAV-derived therapeutics Glybera was approved by the European Medicines Agency (EMA) in 2012, there is an increasing number of AAV-based gene augmentation therapies that have been developed and tested for treating incurable genetic diseases. In the subsequent years, the United States Food and Drug Administration (FDA) approved two additional AAV gene therapy products, Luxturna and Zolgensma, to be launched into the market. Recent breakthroughs in genome editing tools and the combined use with AAV vectors have introduced new therapeutic modalities using somatic gene editing strategies. The promising outcomes from preclinical studies have prompted the continuous evolution of AAV-delivered therapeutics and broadened the scope of treatment options for untreatable diseases. Here, we describe the clinical updates of AAV gene therapies and the latest development using AAV to deliver the CRISPR components as gene editing therapeutics. We also discuss the major challenges and safety concerns associated with AAV delivery and CRISPR therapeutics, and highlight the recent achievement and toxicity issues reported from clinical applications.

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