scholarly journals Clonal reconstruction from co-occurrence of vector integration sites allows accurate quantification of expanding clones in vivo

2021 ◽  
Author(s):  
Sebastian Wagner ◽  
Christoph Baldow ◽  
Andrea Calabria ◽  
Laura Rudilosso ◽  
Pierangela Gallina ◽  
...  
Keyword(s):  
Viral Vectors ◽  
R Package ◽  
Repeated Measurements ◽  
Preclinical Model ◽  
Gene Therapies ◽  
Vector Integration ◽  
Reconstruction Methods ◽  
Integration Sites ◽  
Safety Considerations

High transduction rates of viral vectors in gene therapies (GT) and experimental hematopoiesis ensure a high frequency of gene delivery, although multiple integration events can occur in the same cell. Therefore, tracing of integration sites (IS) leads to mis-quantification of the true clonal spectrum and limits safety considerations in GT. Hence, we use correlations between repeated measurements of IS abundances to estimate their mutual similarity and identify clusters of co-occurring IS, for which we assume a clonal origin. We evaluate the performance, robustness and specificity of our methodology using clonal simulations. The reconstruction methods, implemented and provided as an R-package, are further applied to experimental clonal mixes and to a preclinical model of hematopoietic GT. Our results demonstrate that clonal reconstruction from IS data allows to overcome systematic biases in the clonal quantification as an essential prerequisite for the assessment of safety and long-term efficacy of GT involving integrative vectors.

scholarly journals DNA Palindromes with a Modest Arm Length of ≳20 Base Pairs Are a Significant Target for Recombinant Adeno-Associated Virus Vector Integration in the Liver, Muscles, and Heart in Mice

2007 ◽  
Vol 81 (20) ◽  
pp. 11290-11303 ◽  
Author(s):  
Katsuya Inagaki ◽  
Susanna M. Lewis ◽  
Xiaolin Wu ◽  
Congrong Ma ◽  
David J. Munroe ◽  
...  
Keyword(s):  
Mouse Liver ◽  
Integration Site ◽  
Marker Gene ◽  
Cpg Islands ◽  
Previous Method ◽  
Adeno Associated Virus ◽  
Transcription Start Sites ◽  
Vector Integration ◽  
Integration Sites

ABSTRACT Our previous study has shown that recombinant adeno-associated virus (rAAV) vector integrates preferentially in genes, near transcription start sites and CpG islands in mouse liver (H. Nakai, X. Wu, S. Fuess, T. A. Storm, D. Munroe, E. Montini, S. M. Burgess, M. Grompe, and M. A. Kay, J. Virol. 79:3606-3614, 2005). However, the previous method relied on in vivo selection of rAAV integrants and could be employed for the liver but not for other tissues. Here, we describe a novel method for high-throughput rAAV integration site analysis that does not rely on marker gene expression, selection, or cell division, and therefore it can identify rAAV integration sites in nondividing cells without cell manipulations. Using this new method, we identified and characterized a total of 997 rAAV integration sites in mouse liver, skeletal muscle, and heart, transduced with rAAV2 or rAAV8 vector. The results support our previous observations, but notably they have revealed that DNA palindromes with an arm length of ≳20 bp (total length, ≳40 bp) are a significant target for rAAV integration. Up to ∼30% of total integration events occurred in the vicinity of DNA palindromes with an arm length of ≳20 bp. Considering that DNA palindromes may constitute fragile genomic sites, our results support the notion that rAAV integrates at chromosomal sites susceptible to breakage or preexisting breakage sites. The use of rAAV to label fragile genomic sites may provide an important new tool for probing the intrinsic source of ongoing genomic instability in various tissues in animals, studying DNA palindrome metabolism in vivo, and understanding their possible contributions to carcinogenesis and aging.

scholarly journals 532. Foamy Viral Vector Integration Sites in SCID-Repopulating Cells After MGMTP140K-Mediated In Vivo Selection

2015 ◽  
Vol 23 ◽  
pp. S213-S214
Author(s):  
Miles E. Olszko ◽  
Jennifer E. Adair ◽  
Ian Linde ◽  
Dustin T. Rae ◽  
Patty Trobridge ◽  
...  
Keyword(s):  
Viral Vector ◽  
Vector Integration ◽  
In Vivo Selection ◽  
Integration Sites

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 Polyethyleneimine-Based Lipopolyplexes as Carriers in Anticancer Gene Therapies

Materials ◽  
2021 ◽  
Vol 15 (1) ◽  
pp. 179
Author(s):  
Julia Jerzykiewicz ◽  
Aleksander Czogalla
Keyword(s):  
Nucleic Acid ◽  
Cancer Treatment ◽  
Future Development ◽  
Viral Vectors ◽  
Lipid Vesicles ◽  
Therapeutic Strategies ◽  
In Vivo Studies ◽  
Great Promise ◽  
Gene Therapies

Recent years have witnessed rapidly growing interest in application of gene therapies for cancer treatment. However, this strategy requires nucleic acid carriers that are both effective and safe. In this context, non-viral vectors have advantages over their viral counterparts. In particular, lipopolyplexes—nanocomplexes consisting of nucleic acids condensed with polyvalent molecules and enclosed in lipid vesicles—currently offer great promise. In this article, we briefly review the major aspects of developing such non-viral vectors based on polyethyleneimine and outline their properties in light of anticancer therapeutic strategies. Finally, examples of current in vivo studies involving such lipopolyplexes and possibilities for their future development are presented.

scholarly journals A Mixed-Surface Polyamidoamine Dendrimer for In Vitro and In Vivo Delivery of Large Plasmids

Pharmaceutics ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 619
Author(s):  
Bhairavi Srinageshwar ◽  
Maria Florendo ◽  
Brittany Clark ◽  
Kayla Johnson ◽  
Nikolas Munro ◽  
...  
Keyword(s):  
Viral Vectors ◽  
Viral Vector ◽  
Direct Injection ◽  
Gene Therapies ◽  
Large Plasmids ◽  
Surface Modified ◽  
The Brain ◽  
In Vivo Delivery

Drug delivery to the brain is highly hindered by the presence of the blood–brain barrier (BBB), which prevents the entry of many potential drugs/biomolecules into the brain. One of the current strategies to achieve gene therapy for neurodegenerative diseases involves direct injection of a viral vector into the brain. There are various disadvantages of viral vectors, including limitations of cargo size and safety concerns. Nanomolecules, such as dendrimers, serve as an excellent alternative to viral delivery. In this study, as proof-of-concept, we used a surface-modified dendrimer complex and delivered large plasmids to cells in vitro and in vivo in healthy rats via intracranial injection. The dendrimers were biodegradable by chemicals found within cells and toxicity assays revealed that the modified dendrimers were much less toxic than unmodified amine-surface dendrimers. As mentioned in our previous publication, these dendrimers with appropriately modified surfaces are safe, can deliver large plasmids to the brain, and can overcome the cargo size limitations associated with viral vectors. The biocompatibility of this dendritic nanomolecule and the ability to finely tune its surface chemistry provides a gene delivery system that could facilitate future in vivo cellular reprograming and other gene therapies.

scholarly journals Foamy viral vector integration sites in SCID-repopulating cells after MGMTP140K-mediated in vivo selection

Gene Therapy ◽  
2015 ◽  
Vol 22 (7) ◽  
pp. 591-595 ◽  
Author(s):  
M E Olszko ◽  
J E Adair ◽  
I Linde ◽  
D T Rae ◽  
P Trobridge ◽  
...  
Keyword(s):  
Viral Vector ◽  
Vector Integration ◽  
In Vivo Selection ◽  
Integration Sites

scholarly journals Safety considerations for nanoparticle gene delivery in pediatric brain tumors

Nanomedicine ◽  
2020 ◽  
Vol 15 (18) ◽  
pp. 1805-1815
Author(s):  
Kathryn M Luly ◽  
John Choi ◽  
Yuan Rui ◽  
Jordan J Green ◽  
Eric M Jackson
Keyword(s):  
Gene Delivery ◽  
Viral Vectors ◽  
Pediatric Brain Tumor ◽  
Standard Of Care ◽  
Pediatric Brain Tumors ◽  
Current Standard ◽  
Gene Therapies ◽  
Safety Considerations ◽  
Tumor Types ◽  
Pediatric Brain

Current standard of care for many CNS tumors involves surgical resection followed by chemotherapy and/or radiation. Some pediatric brain tumor types are infiltrative and diffuse in nature, which reduces the role for surgery. Furthermore, children are extremely vulnerable to neurological sequelae from surgery and radiation therapy, thus alternative approaches are in critical need. As molecular targets underlying various cancers become more clearly defined, there is an increasing push for targeted gene therapies. Viral vectors and nonviral nanoparticles have been thoroughly investigated for gene delivery and show promise as vectors for gene therapy for pediatric brain cancer. Here, we review inorganic and organic materials in development for nanoparticle gene delivery to the brain with a particular focus on safety.

scholarly journals Quantifying CRISPR off-target effects

2019 ◽  
Vol 3 (3) ◽  
pp. 327-334 ◽  
Author(s):  
Soragia Athina Gkazi
Keyword(s):  
Genome Editing ◽  
Viral Vectors ◽  
Insertional Mutagenesis ◽  
Severe Combined Immunodeficiency ◽  
Gene Therapies ◽  
Genome Wide ◽  
Large Indels ◽  
Target Effects

Abstract Recent advances in the era of genetic engineering have significantly improved our ability to make precise changes in the genomes of human cells. Throughout the years, clinical trials based on gene therapies have led to the cure of diseases such as X-linked severe combined immunodeficiency (SCID-X1), adenosine deaminase deficiency (ADA-SCID) and Wiskott–Aldrich syndrome. Despite the success gene therapy has had, there is still the risk of genotoxicity due to the potential oncogenesis introduced by utilising viral vectors. Research has focused on alternative strategies like genome editing without viral vectors as a means to reduce genotoxicity introduced by the viral vectors. Although there is an extensive use of RNA-guided genome editing via the clustered regularly interspaced short palindromic repeats (CRISPR) and associated protein-9 (Cas9) technology for biomedical research, its genome-wide target specificity and its genotoxic side effects remain controversial. There have been reports of on- and off-target effects created by CRISPR–Cas9 that can include small and large indels and inversions, highlighting the potential risk of insertional mutagenesis. In the last few years, a plethora of in silico, in vitro and in vivo genome-wide assays have been introduced with the sole purpose of profiling these effects. Here, we are going to discuss the genotoxic obstacles in gene therapies and give an up-to-date overview of methodologies for quantifying CRISPR–Cas9 effects.

scholarly journals In vivo directed evolution of AAV in the primate retina

2019 ◽  
Author(s):  
Leah C. Byrne ◽  
Timothy P. Day ◽  
Meike Visel ◽  
Cecile Fortuny ◽  
Deniz Dalkara ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Directed Evolution ◽  
Viral Vectors ◽  
Outer Retina ◽  
Library Administration ◽  
Viral Capsids ◽  
High Performing ◽  
Gene Therapies ◽  
In Trans

AbstractEfficient AAV-mediated gene delivery remains a significant obstacle to effective retinal gene therapies. Here, we apply directed evolution – guided by deep sequencing and followed by direct in vivo secondary selection of high-performing vectors with a GFP-barcoded library – to create AAV viral capsids with new capabilities to deliver genes to the outer retina in primates. A replication incompetent library, produced via providing rep in trans, was created to mitigate risk of AAV propagation. Six rounds of in vivo selection with this library in primates – involving intravitreal library administration, recovery of genomes from outer retina, and extensive next generation sequencing of each round – resulted in vectors with redirected tropism to the outer retina and increased gene delivery efficiency to retinal cells. These new viral vectors expand the toolbox of vectors available for primate retina, and may enable less invasive delivery of therapeutic genes to patients, potentially offering retina-wide infection at a similar dosage to vectors currently in clinical use.

scholarly journals Riboswitches for Controlled Expression of Therapeutic Transgenes Delivered by Adeno-Associated Viral Vectors

2021 ◽  
Vol 14 (6) ◽  
pp. 554
Author(s):  
Zachary J. Tickner ◽  
Michael Farzan
Keyword(s):  
Gene Therapy ◽  
Animal Models ◽  
Mammalian Cells ◽  
Viral Vectors ◽  
Adeno Associated Virus ◽  
Gene Therapies ◽  
Expression Control ◽  
Transgene Delivery ◽  
Improved Performance

Vectors developed from adeno-associated virus (AAV) are powerful tools for in vivo transgene delivery in both humans and animal models, and several AAV-delivered gene therapies are currently approved for clinical use. However, AAV-mediated gene therapy still faces several challenges, including limited vector packaging capacity and the need for a safe, effective method for controlling transgene expression during and after delivery. Riboswitches, RNA elements which control gene expression in response to ligand binding, are attractive candidates for regulating expression of AAV-delivered transgene therapeutics because of their small genomic footprints and non-immunogenicity compared to protein-based expression control systems. In addition, the ligand-sensing aptamer domains of many riboswitches can be exchanged in a modular fashion to allow regulation by a variety of small molecules, proteins, and oligonucleotides. Riboswitches have been used to regulate AAV-delivered transgene therapeutics in animal models, and recently developed screening and selection methods allow rapid isolation of riboswitches with novel ligands and improved performance in mammalian cells. This review discusses the advantages of riboswitches in the context of AAV-delivered gene therapy, the subsets of riboswitch mechanisms which have been shown to function in human cells and animal models, recent progress in riboswitch isolation and optimization, and several examples of AAV-delivered therapeutic systems which might be improved by riboswitch regulation.

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