Non-Integrating Lentiviral Vectors in Clinical Applications: A Glance Through

Lentiviral vectors (LVs) play an important role in gene therapy and have proven successful in clinical trials. LVs are capable of integrating specific genetic materials into the target cells and allow for long-term expression of the cDNA of interest. The use of non-integrating LVs (NILVs) reduces insertional mutagenesis and the risk of malignant cell transformation over integrating lentiviral vectors. NILVs enable transient expression or sustained episomal expression, especially in non-dividing cells. Important modifications have been made to the basic human immunodeficiency virus (HIV) structures to improve the safety and efficacy of LVs. NILV-aided transient expression has led to more pre-clinical studies on primary immunodeficiencies, cytotoxic cancer therapies, and hemoglobinopathies. Recently, the third generation of self-inactivating LVs was applied in clinical trials for recombinant protein production, vaccines, gene therapy, cell imaging, and induced pluripotent stem cell (iPSC) generation. This review discusses the basic lentiviral biology and the four systems used for generating NILV designs. Mutations or modifications in LVs and their safety are addressed with reference to pre-clinical studies. The detailed application of NILVs in promising pre-clinical studies is also discussed.

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Lentiviral Vectors for the Treatment and Prevention of Cystic Fibrosis Lung Disease

Genes ◽

10.3390/genes10030218 ◽

2019 ◽

Vol 10 (3) ◽

pp. 218 ◽

Cited By ~ 16

Author(s):

Laura Marquez Loza ◽

Eric Yuen ◽

Paul McCray

Keyword(s):

Cystic Fibrosis ◽

Gene Therapy ◽

Clinical Trials ◽

Insertional Mutagenesis ◽

Primary Cultures ◽

Severe Combined Immunodeficiency ◽

Lentiviral Vectors ◽

Retroviral Vectors ◽

Airway Epithelia

Despite the continued development of cystic fibrosis transmembrane conductance regulator (CFTR) modulator drugs for the treatment of cystic fibrosis (CF), the need for mutation agnostic treatments remains. In a sub-group of CF individuals with mutations that may not respond to modulators, such as those with nonsense mutations, CFTR gene transfer to airway epithelia offers the potential for an effective treatment. Lentiviral vectors are well-suited for this purpose because they transduce nondividing cells, and provide long-term transgene expression. Studies in primary cultures of human CF airway epithelia and CF animal models demonstrate the long-term correction of CF phenotypes and low immunogenicity using lentiviral vectors. Further development of CF gene therapy requires the investigation of optimal CFTR expression in the airways. Lentiviral vectors with improved safety features have minimized insertional mutagenesis safety concerns raised in early clinical trials for severe combined immunodeficiency using γ-retroviral vectors. Recent clinical trials using improved lentiviral vectors support the feasibility and safety of lentiviral gene therapy for monogenetic diseases. While work remains to be done before CF gene therapy reaches the bedside, recent advances in lentiviral vector development reviewed here are encouraging and suggest it could be tested in clinical studies in the near future.

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New Molecular Surrogate Assay for Genotoxicity Assessment of Gene Therapy Vectors (SAGA)

Blood ◽

10.1182/blood.v128.22.4710.4710 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4710-4710 ◽

Cited By ~ 1

Author(s):

Adrian Schwarzer ◽

Steven R Talbot ◽

Oliver Dittrich-Breiholz ◽

Adrian J Thrasher ◽

Bobby Gaspar ◽

...

Keyword(s):

Gene Therapy ◽

Clinical Trials ◽

Insertional Mutagenesis ◽

Lentiviral Vectors ◽

Mutagenic Potential ◽

In Vivo Models ◽

Core Set ◽

Gene Therapy Vectors ◽

Surrogate Assay

Abstract The variety of gene therapy vectors for a multitude of different diseases has increased tremendously over the years. However, a number of patients that underwent gene therapy in different trials developed hematological malignancy caused by integration of the provirus in the vicinity of proto-oncogenes. These severe adverse advents prompted intense research efforts towards safer gene therapy, leading to the removal of the long terminal repeat enhancer elements and the use of internal promoters in retroviral vectors. Still, a bottleneck of transition from basic research to clinical application is the test for safety of integrating retro- and lentiviral vectors. Instead of laborious in vivo models with limited predictive value, in vitro assays to screen for insertional mutagenesis are strongly desirable. A decade ago, our lab developed the in vitro immortalization (IVIM) assay to quantify the genotoxic potential of viral vectors, which has been widely used to complete preclinical safety documentation of newly developed integrating vector systems. Despite general acceptance in the field of hematopoietic gene therapy, bias for insertional mutants of the myeloid lineage, a low sensitivity and a long assay run time are clear limitations. We now developed the molecular surrogate assay for genotoxicity assessment (SAGA). The new test is more robust, sensitive and biologically informative. As input we used murine lineage-negative hematopoietic stem and progenitor cells (HSPC) that were cultured as described for the IVIM assay. The murine HSPC were transduced with a number of different gammaretro- and lentiviral vectors, including vectors that have been employed in clinical trials for X-SCID and Wiskott-Aldrich Syndrome. After 14 days, whole mRNA was isolated from transduced and non-transduced samples and analyzed by Agilent custom microarrays (n=86) and qPCR from nine independent SAGA assays. We applied several Machine Learning algorithms to derive a core set of genes which distinguishes transformed from non-transformed samples in each individual SAGA assay. This set of genes from the individual analysis was further analyzed to derive a core set of genes that is able to robustly separate transformed from non-transformed samples in all assays performed. In order to account for platform-specific effects we validated all microarray results by conventional qPCR-methodology. The SAGA gene set was then cross-validated in an independent validation cohort of SAGA-assays that were not part of the SAGA-training set from which the signature was derived from. The SAGA assay was used to quantify the mutagenic potential of several benchmark vectors. It correctly assigned a high mutagenic potential to vectors (MFG.yc and CMMP.WASP) which led to serious adverse events (SAEs) in clinical trials. Most importantly, the SAGA assay reliably scored high for mutagenic vectors, even when the vector did not transform in IVIM-assays conducted in parallel, demonstrating the higher sensitivity of the SAGA-principle. In contrast, SIN lentiviral vectors with weaker internal promoters (LV.EFS.yc and LV.EFS.ADA) showed no enrichment of the SAGA-core signature and hence scored much safer in the SAGA test. We present the results for these vectors side-by-side either using IVIM or SAGA. In summary, we generated an advanced version of the currently used in vitro insertional mutagenesis screening system by integrating a molecular read-out which enhances reproducibility, sensitivity and reduces assay duration, paving the way for a better preclinical risk assessment of gene therapy vectors. Disclosures No relevant conflicts of interest to declare.

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Ex Vivo Expansion of Retrovirally-Transduced Primate CD34+ Cells Results in Preferential Engraftment and Persistence of Clones with MDS1/EVI1 Insertion Sites.

Blood ◽

10.1182/blood.v108.11.203.203 ◽

2006 ◽

Vol 108 (11) ◽

pp. 203-203

Author(s):

Theo Gomes ◽

Stephanie Sellers ◽

Robert E. Donahue ◽

Rima Adler ◽

Andre La Rochelle ◽

...

Keyword(s):

Gene Therapy ◽

Insertional Mutagenesis ◽

Ex Vivo ◽

Safety Issue ◽

Retroviral Vectors ◽

Ex Vivo Expansion ◽

Target Cells ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Clinical Gene Therapy

Abstract There is increasing evidence that insertional activation of proto-oncogenes by retroviral vectors is a significant safety issue that must be addressed before clinical gene therapy, particularly targeting hematopoietic stem and progenitor cells, can be further developed. The risk of insertional mutagenesis for replication-incompetent retroviral vectors has been assumed to be low until the occurence of T cell leukemias in children treated with HSC-directed gene therapy for X-SCID, and recent evidence that retroviral integration is more common in the promoter region of transcriptionally-active genes. The occurence of “common integration sites” in a particular gene also suggests a non-random insertion pattern, and/or immortalization or other change in the behavior of a clone harboring an insertion in these particular genes. We have previously reported a highly non-random occurence of 14 unique vector integrations in the first two introns of the MDS1/EVI1 proto-oncogene out of a total of 702 identified from myeloid cells of 9 rhesus macaques at least 6 months post-transplantion of retrovirally-transduced CD34+ cells.(Calmels et al, 2005). This same gene locus was found frequently activated by insertions in murine bone marrow cells immortalized in long-term in vitro culture after transduction with retroviral vectors.(Du et al Blood, 2005) To begin to investigate the factors contributing to this worrisome finding, particularly given the very recent report of a marked over-representation of MDS1/EVI1 insertions in a human clinical gene therapy trial for chronic granulomatous disease, we asked whether continued ex vivo expansion of transduced CD34+ cells prior to transplantation would further select for clones with insertions in MDS1/EVI1 or other proto-oncogenes. Rhesus CD34+ cells were transduced with the G1Na standard retroviral vector, identical to that used in the prior studies, using our standard 96 hour transduction protocol in the presence of Retronectin and SCF, FLT3L and thrombopoietin. At the end of transduction, all cells were continued in culture for an additional 7 days under the same culture conditions, and then reinfused into the donor animal following 1200 rads TBI. At 1 month post-transplant there were no CIS and no MDS1/EVI1 insertions identified. However, at 6 months post-transplantation 5 out of 27 (19%) of the unique insertions identified in granulocytes were within the first two introns of MDS1/EVI1, very significantly higher than the 2% of MDS1/EVI1 insertions (14 of 702) identified in animals that were transplanted with cells not subjected to additional ex vivo expansion.(p<.0001) One MDS1/EVI1 clone constituted 14% of overall sequences identified, and the 5 clones constituted 37% of total sequences identified. This strongly suggests that the over-representation of this locus in engrafting cells is due to a potent immortalizing signal provided by activation of the MDS1/EVI1 gene products by the stonger retroviral promoter/enhancer, and that the need for extended ex vivo culture of target cells may select for insertion events activating this locus. It also suggests that strategies involving prolonged ex vivo expansion or selection of transduced cells could increase the risk of gene therapy utilizing integrating vectors targeting primitive hematopoietic cells.

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A guide in lentiviral vector production for hard-to-transfect cells, using cardiac-derived c-kit expressing cells as a model system

Scientific Reports ◽

10.1038/s41598-021-98657-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

V. Kalidasan ◽

Wai Hoe Ng ◽

Oluwaseun Ayodeji Ishola ◽

Nithya Ravichantar ◽

Jun Jie Tan ◽

...

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Gene Delivery ◽

Delivery System ◽

Lentiviral Vector ◽

Lentiviral Vectors ◽

Model System ◽

Selection Marker ◽

Target Cells ◽

Primary Cells

AbstractGene therapy revolves around modifying genetic makeup by inserting foreign nucleic acids into targeted cells via gene delivery methods to treat a particular disease. While the genes targeted play a key role in gene therapy, the gene delivery system used is also of utmost importance as it determines the success of gene therapy. As primary cells and stem cells are often the target cells for gene therapy in clinical trials, the delivery system would need to be robust, and viral-based entries such as lentiviral vectors work best at transporting the transgene into the cells. However, even within lentiviral vectors, several parameters can affect the functionality of the delivery system. Using cardiac-derived c-kit expressing cells (CCs) as a model system, this study aims to optimize lentiviral production by investigating various experimental factors such as the generation of the lentiviral system, concentration method, and type of selection marker. Our findings showed that the 2nd generation system with pCMV-dR8.2 dvpr as the packaging plasmid produced a 7.3-fold higher yield of lentiviral production compared to psPAX2. Concentrating the virus with ultracentrifuge produced a higher viral titer at greater than 5 × 105 infectious unit values/ml (IFU/ml). And lastly, the minimum inhibitory concentration (MIC) of puromycin selection marker was 10 μg/mL and 7 μg/mL for HEK293T and CCs, demonstrating the suitability of antibiotic selection for all cell types. This encouraging data can be extrapolated and applied to other difficult-to-transfect cells, such as different types of stem cells or primary cells.

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Gene therapy for PNP deficiency protocol

LymphoSign Journal ◽

10.14785/lymphosign-2018-0007 ◽

2018 ◽

Vol 5 (3) ◽

pp. 115-120

Author(s):

Linda Vong

Keyword(s):

Gene Therapy ◽

Stem Cell ◽

Insertional Mutagenesis ◽

First Generation ◽

Therapeutic Option ◽

Lentiviral Vectors ◽

Retroviral Vectors ◽

Post Transplantation ◽

Hematopoietic Stem ◽

Key Enzyme

Purine nucleoside phosphorylase (PNP) is a key enzyme required for the degradation of purine nucleosides into uric acid or their salvage into nucleic acids. Patients who are deficient in PNP suffer from progressive T cell immunodeficiency, with increased susceptibility to infections, autoimmunity, and neurologic abnormalities. In the absence of successful treatment to restore immune function, these patients rarely survive to adulthood. Hematopoietic stem cell transplantation is the only known cure for PNP deficiency. Use of an HLA-matched donor is preferable as the outcome with alternative donors have been variable; however, this option is rarely available. Gene therapy represents a therapeutic option that bypasses the need for a donor, and thus associated complications. Although first generation γ-retroviral vectors have been successful in some immunodeficiencies, in others, evidence of insertional mutagenesis prompted a halt in their use. More recently, the introduction of safer lentiviral vectors holds promise in offering a viable option to treat immunodeficiency. Here, we present a clinical trial protocol utilizing self-inactivating lentiviral vectors to treat PNP deficiency. Patients will be evaluated up to 3 years post-transplantation to determine the safety of lentiviral-treated stem cell infusion, as well as the extent of immune reconstitution. Statement of novelty: This protocol describes the novel treatment of PNP deficiency using lentiviral-based gene therapy.

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The Old and the New: Prospects for Non-Integrating Lentiviral Vector Technology

Viruses ◽

10.3390/v12101103 ◽

2020 ◽

Vol 12 (10) ◽

pp. 1103 ◽

Cited By ~ 1

Author(s):

Luis Apolonia

Keyword(s):

Insertional Mutagenesis ◽

Lentiviral Vector ◽

Genetic Material ◽

Lentiviral Vectors ◽

Cell Types ◽

Target Cells ◽

Attachment Sites ◽

Dividing Cells ◽

Multiple Cell ◽

Episomal Dna

Lentiviral vectors have been developed and used in multiple gene and cell therapy applications. One of their main advantages over other vectors is the ability to integrate the genetic material into the genome of the host. However, this can also be a disadvantage as it may lead to insertional mutagenesis. To address this, non-integrating lentiviral vectors (NILVs) were developed. To generate NILVs, it is possible to introduce mutations in the viral enzyme integrase and/or mutations on the viral DNA recognised by integrase (the attachment sites). NILVs are able to stably express transgenes from episomal DNA in non-dividing cells or transiently if the target cells divide. It has been shown that these vectors are able to transduce multiple cell types and tissues. These characteristics make NILVs ideal vectors to use in vaccination and immunotherapies, among other applications. They also open future prospects for NILVs as tools for the delivery of CRISPR/Cas9 components, a recent revolutionary technology now widely used for gene editing and repair.

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IL-7 surface-engineered lentiviral vectors promote survival and efficient gene transfer in resting primary T lymphocytes

Blood ◽

10.1182/blood-2002-07-2224 ◽

2003 ◽

Vol 101 (6) ◽

pp. 2167-2174 ◽

Cited By ~ 83

Author(s):

Els Verhoeyen ◽

Valerie Dardalhon ◽

Odile Ducrey-Rundquist ◽

Didier Trono ◽

Naomi Taylor ◽

...

Keyword(s):

Gene Therapy ◽

T Cells ◽

T Cell ◽

Gene Transfer ◽

Lentiviral Vectors ◽

Target Cells ◽

Interleukin 7 ◽

Efficient Gene ◽

Cognate Antigen ◽

Therapy Target

Important gene therapy target cells such as resting human T cells are refractory to transduction with lentiviral vectors. Completion of reverse transcription, nuclear import, and subsequent integration of the lentiviral genome occur in these cells only if they have been activated. In T-cell–based gene therapy trials performed to date, cells have been activated via their cognate antigen receptor. To couple activation with gene transfer, we previously generated lentiviral vectors displaying an anti-CD3 scFv fragment that allowed up to 48% transduction of freshly isolated T cells. However, transduction of highly purified resting T cells with these anti-CD3–displaying lentiviral vectors was inefficient and shifted the T cells from the naive to the memory phenotype. Here, we describe interleukin-7 (IL-7)–displaying HIV-1–derived vectors. Like recombinant IL-7, these modified particles could promote the survival of primary T cells placed in culture without inducing a naive-to-memory phenotypic switch. Furthermore, a single exposure to the IL-7–displaying vectors resulted in efficient gene transfer in both resting memory adult T cells and naive cord blood T cells. With adult naive T cells, preactivation with recombinant IL-7 was necessary for efficient gene transfer. Altogether, these results suggest that IL-7–displaying vectors could constitute interesting tools for T-cell–targeted gene therapy.

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Implementing the human artificial chromosome gene therapy platform remains challenging, but continuous animal model research will advance the platform closer to clinical trials

10.31219/osf.io/a53f7 ◽

2021 ◽

Author(s):

Moataz Dowaidar

Keyword(s):

Gene Therapy ◽

Clinical Trials ◽

Animal Model ◽

Human Artificial Chromosome ◽

Target Cells ◽

Minimal Risk ◽

Artificial Chromosomes ◽

Model Research ◽

Alphoid Dna

A normal degree of ectopic gene expression, infinite retention in target cells without chromosomal integration, minimal risk of cell or neoplastic transformation, and minimal or no immunogenicity are all critical characteristics for vectors employed in gene therapy. HACs were produced and used as autonomous vectors to compensate for genetic defects in mouse and human cell cultures. Bottom-up human artificial chromosomes (HACs) were studied for functional transgene expression in vitro and in vivo mice models. The primary advantages of synthesized alphoid-HACs over top-down HACs are their defined and documented structure, as well as their relative simplicity of modification in adding numerous Cre-lox-type transgen loading sites. The HAC transfer method's efficacy has greatly increased in recent years. Despite significant progress in developing alphoid-HAC-based gene therapy models, the technology still has a number of drawbacks, including low HAC efficiency, complex repeated HAC alphoid-DNA structure, large DNA fragmentation difficulties outside eukaryotic cells, inefficient transfer of chromosomes to target cells, and variable mitotic stability. The quantity and quality of PSC-derived or reversibly immortalized stem/precursor cells that can transplant specific tissues are also critical determinants in the effectiveness of HAC-based tissue replacement therapies. Translating the HAC-based gene therapy platform remains difficult, but ongoing animal model research will move the HAC platform closer to clinical trials.

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TREATMENT OF HIV-INFECTION BY MEANS OF GENE THERAPY

Annals of the Russian academy of medical sciences ◽

10.15690/vramn.v67i5.268 ◽

2012 ◽

Vol 67 (5) ◽

pp. 16-23 ◽

Cited By ~ 1

Author(s):

D. V. Glazkova ◽

E. V. Bogoslovskaya ◽

M. L. Markelov ◽

G. A. Shipulin ◽

V. V. Pokrovskii

Keyword(s):

Gene Therapy ◽

Clinical Trials ◽

Antiretroviral Therapy ◽

Hiv Infection ◽

Mechanisms Of Action ◽

Hiv Treatment ◽

Target Cells ◽

New Approach ◽

Recent Advances ◽

Antiviral Genes

Current methods of HIV treatment can contain a progression of the disease; however they do not lead to a cure. Lifelong antiretroviral therapy is therefore necessary, leading to problems of cost and toxicity of chemical drugs. The recent advances in science have allowed a new approach to the HIV-treatment — gene therapy. In the present publication we focus on one strategy of the gene therapy called «intracellular immunization». The strategy is based on the introducing of antiviral genes into the HIV-sensitive cells. We highlight the mechanisms of action of various antiviral genetic agents and discuss some issues concerning target cells and genes delivery. Finally we summarize the results of certain gene therapy clinical trials.

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Preclinical and clinical advances in transposon-based gene therapy

Bioscience Reports ◽

10.1042/bsr20160614 ◽

2017 ◽

Vol 37 (6) ◽

Cited By ~ 24

Author(s):

Jaitip Tipanee ◽

Yoke Chin Chai ◽

Thierry VandenDriessche ◽

Marinee K. Chuah

Keyword(s):

Gene Therapy ◽

Clinical Trials ◽

Viral Vectors ◽

Ex Vivo ◽

Cell Types ◽

Sleeping Beauty ◽

Target Cells ◽

Hematologic Cancer ◽

Clinical Advances

Transposons derived from Sleeping Beauty (SB), piggyBac (PB), or Tol2 typically require cotransfection of transposon DNA with a transposase either as an expression plasmid or mRNA. Consequently, this results in genomic integration of the potentially therapeutic gene into chromosomes of the desired target cells, and thus conferring stable expression. Non-viral transfection methods are typically preferred to deliver the transposon components into the target cells. However, these methods do not match the efficacy typically attained with viral vectors and are sometimes associated with cellular toxicity evoked by the DNA itself. In recent years, the overall transposition efficacy has gradually increased by codon optimization of the transposase, generation of hyperactive transposases, and/or introduction of specific mutations in the transposon terminal repeats. Their versatility enabled the stable genetic engineering in many different primary cell types, including stem/progenitor cells and differentiated cell types. This prompted numerous preclinical proof-of-concept studies in disease models that demonstrated the potential of DNA transposons for ex vivo and in vivo gene therapy. One of the merits of transposon systems relates to their ability to deliver relatively large therapeutic transgenes that cannot readily be accommodated in viral vectors such as full-length dystrophin cDNA. These emerging insights paved the way toward the first transposon-based phase I/II clinical trials to treat hematologic cancer and other diseases. Though encouraging results were obtained, controlled pivotal clinical trials are needed to corroborate the efficacy and safety of transposon-based therapies.

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