scholarly journals HIV-1 sequences in lentiviral vector genomes can be substantially reduced without compromising transduction efficiency

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Helin Sertkaya ◽  
Mattia Ficarelli ◽  
Nathan P. Sweeney ◽  
Hannah Parker ◽  
Conrad A. Vink ◽  
...  
Keyword(s):  
Lentiviral Vector ◽  
Lentiviral Vectors ◽  
Large Deletion ◽  
Transduction Efficiency ◽  
Rev Response Element ◽  
Vector Genome ◽  
Long Read ◽  
Viral Sequences ◽  
Hiv 1 ◽  
Splice Site Usage

AbstractMany lentiviral vectors used for gene therapy are derived from HIV-1. An optimal vector genome would include only the viral sequences required for transduction efficiency and gene expression to minimize the amount of foreign sequence inserted into a patient’s genome. However, it remains unclear whether all of the HIV-1 sequence in vector genomes is essential. To determine which viral sequences are required, we performed a systematic deletion analysis, which showed that most of the gag region and over 50% of the env region could be deleted. Because the splicing profile for lentiviral vectors is poorly characterized, we used long-read sequencing to determine canonical and cryptic splice site usage. Deleting specific regions of env sequence reduced the number of splicing events per transcript and increased the proportion of unspliced genomes. Finally, combining a large deletion in gag with repositioning the Rev-response element downstream of the 3’ R to prevent its reverse transcription showed that 1201 nucleotides of HIV-1 sequence can be removed from the integrated vector genome without substantially compromising transduction efficiency. Overall, this allows the creation of lentiviral vector genomes that contain minimal HIV-1 sequence, which could improve safety and transfer less viral sequence into a patient’s DNA.

HIV-1 sequences in lentiviral vector genomes can be substantially reduced without compromising transduction efficiency

2021 ◽  
Author(s):  
Helin Sertkaya ◽  
Mattia Ficarelli ◽  
Nathan P Sweeney ◽  
Hannah Parker ◽  
Conrad A Vink ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Lentiviral Vector ◽  
Maximum Size ◽  
Transduction Efficiency ◽  
Genome Expression ◽  
Vector Genome ◽  
Oxford Nanopore ◽  
Functional Relevance ◽  
Viral Sequences ◽  
Hiv 1

Abstract Many lentiviral vector genomes used for gene therapy are derived from HIV-1 and contain sequences required for genome expression, encapsidation and reverse transcription. In addition, they contain substantial regions of gag and env. Extraneous viral sequence in the genome reduces the maximum size of the transgene expression cassette and potentially increases biosafety concerns. Importantly, it remains unclear whether all of the HIV-1 sequence is required for transduction efficiency. To determine the functional relevance of the viral sequences in the vector genome, we performed a deletion analysis. Most of gag could be deleted without compromising vector titre. Within env, only the RRE is necessary, allowing over 50% of this region to be deleted. Oxford Nanopore sequencing showed that deleting 507 nt of env decreased the number of pre-mRNA splicing events per transcript and increased the proportion of unspliced genomes. Finally, combining the deletion in gag with moving the RRE downstream of the 3’ R to prevent its reverse transcription showed that substantial amounts of HIV-1 sequence can be removed from the vector genome without compromising transduction efficiency. Overall, this allows the creation of lentiviral vector genomes that contain minimal HIV-1 sequence, which could improve biosafety and increase the transgene capacity.

scholarly journals Lentiviral Vector-Based Prime/Boost Vaccination against AIDS: Pilot Study Shows Protection against Simian Immunodeficiency Virus SIVmac251 Challenge in Macaques

2009 ◽  
Vol 83 (21) ◽  
pp. 10963-10974 ◽  
Author(s):  
Anne-Sophie Beignon ◽  
Karine Mollier ◽  
Christelle Liard ◽  
Frédéric Coutant ◽  
Sandie Munier ◽  
...  
Keyword(s):  
T Cells ◽  
Pilot Study ◽  
Immune Responses ◽  
Simian Immunodeficiency Virus ◽  
Lentiviral Vector ◽  
Lentiviral Vectors ◽  
Cellular Immune Responses ◽  
Immunodeficiency Virus ◽  
Cellular Immune ◽  
Hiv 1

ABSTRACT AIDS vaccination has a pressing need for more potent vaccination vectors capable of eliciting strong, diversified, and long-lasting cellular immune responses against human immunodeficiency virus (HIV). Lentiviral vectors have demonstrated efficiency not only as gene delivery vehicles for gene therapy applications but also as vaccination tools. This is likely due to their ability to transduce nondividing cells, including dendritic cells, enabling sustained endogenous antigen presentation and thus the induction of high proportions of specific cytotoxic T cells and long-lasting memory T cells. We show in a first proof-of-concept pilot study that a prime/boost vaccination strategy using lentiviral vectors pseudotyped with a glycoprotein G from two non-cross-reactive vesicular stomatitis virus serotypes elicited robust and broad cellular immune responses against the vector-encoded antigen, simian immunodeficiency virus (SIV) GAG, in cynomolgus macaques. Vaccination conferred strong protection against a massive intrarectal challenge with SIVmac251, as evidenced both by the reduction of viremia at the peak of acute infection (a mean of over 2 log10 fold reduction) and by the full preservation of the CD28+ CD95+ memory CD4+ T cells during the acute phase, a strong correlate of protection against pathogenesis. Although vaccinees continued to display lower viremia than control macaques during the early chronic phase, these differences were not statistically significant by day 50 postchallenge. A not-optimized SIV GAG antigen was chosen to show the strong potential of the lentiviral vector system for vaccination. Given that a stronger protection can be anticipated from a modern HIV-1 antigen design, gene transfer vectors derived from HIV-1 appear as promising candidates for vaccination against HIV-1 infection.

scholarly journals A single administration of lentiviral vectors expressing either full-length human immunodeficiency virus 1 (HIV-1)HXB2 Rev/Env or codon-optimized HIV-1JR-FL gp120 generates durable immune responses in mice

2006 ◽  
Vol 87 (6) ◽  
pp. 1625-1634 ◽  
Author(s):  
Viviana Buffa ◽  
Donatella R. M. Negri ◽  
Pasqualina Leone ◽  
Roberta Bona ◽  
Martina Borghi ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Immune Responses ◽  
Viral Vectors ◽  
Lentiviral Vector ◽  
Lentiviral Vectors ◽  
Full Length ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Human Immunodeficiency Virus 1

Genetic immunization using viral vectors provides an effective means to elicit antigen-specific cellular immune responses. Several viral vectors have proven efficacious in inducing immune responses after direct injection in vivo. Among them, recombinant, self-inactivating lentiviral vectors are very attractive delivery systems, as they are able to efficiently transduce into and express foreign genes in a wide variety of mammalian cells. A self-inactivating lentiviral vector was evaluated for the delivery of human immunodeficiency virus 1 (HIV-1) envelope sequences in mice in order to elicit specific immune responses. With this aim, BALB/c mice were immunized with a single injection of self-inactivating lentiviral vectors carrying either the full-length HIV-1HXB2 Rev/Env (TY2-IIIBEnv) or the codon-optimized HIV-1JR-FL gp120 (TY2-JREnv) coding sequence. Both vectors were able to elicit specific cellular responses efficiently, as measured by gamma interferon ELISPOT and chromium-release assays, upon in vitro stimulation of splenocytes from BALB/c immunized mice. However, only the TY2-JREnv-immunized mice were able to elicit specific humoral responses, measured as anti-gp120 antibody production. These data provide the first evidence that a single, direct, in vivo administration of a lentiviral vector encoding a viral gene might represent a useful strategy for vaccine development.

Diminished Mobilization of Self-Inactivating (SIN) Lentiviral Vectors Containing Globin Regulatory Elements Compared to Those Containing a Retroviral Long Terminal Repeat.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5271-5271
Author(s):  
Hideki Hanawa ◽  
Derek A. Persons ◽  
Takashi Shimada ◽  
Arthur W. Nienhuis
Keyword(s):  
Stem Cell ◽  
Long Terminal Repeat ◽  
Hela Cells ◽  
Lentiviral Vector ◽  
Severe Combined Immunodeficiency ◽  
Lentiviral Vectors ◽  
Regulatory Elements ◽  
Terminal Repeat ◽  
Vector Genome ◽  
The Common

Abstract Stem cell transfer was successful in treating severe combined immunodeficiency due to deficiencies of the common γ-chain of the lymphoid cytokine receptor (N Engl J Med346:1185, 2002) and adenosine deaminase (Science296:2410, 2002) but 2 of 10 children in the common γ-chain trial developed a lymphoproliferative disease secondary in part due to activation of the LMO2 proto-oncogene by the retroviral long terminal repeat (LTR) (Science302:415, 2003). Both oncoretroviral and lentiviral vectors integrate preferentially into transcriptionally active genes so that vector design to improve safety is important. In focusing on the interactions between vector sequences and the genome surrounding integration sites, we have used self-inactivating (SIN) lentiviral vectors with transcriptionally inactive LTRs. One assay detects mobilization of the vector genome by rescue of a GFP marker while a second detects vector encoded tat transcription (Blood102:249a, 2003). Approximately 1 in 1,000 to 1 in 3,000 vector genomes containing the Mouse Stem Cell Virus (MSCV) LTR are transcribed with transcription arising from cryptic promoters within the vector genome or from nearby genomic sequences. The frequency of genome transcription was diminished by removal of the MSCV LTR enhancer or by addition of an insulator element from the β-globin locus control region (LCR) to the lentiviral SIN LTR. We have now evaluated the effect of globin regulatory elements on transcription of integrated SIN lentiviral vector genomes. Initially, we substituted the LTR GFP cassette in vector MSCV-U3 for one in which elements from the β-globin LCR were linked to the β-globin promoter driving GFP in the reverse transcriptional orientation (d432βGFPim). Two additional vectors were also studied; the globin LCR β-promoter GFP cassette was reversed and the globin RNA processing signals removed to derive vector Fd432βGFP and then it was modified by substituting larger LCR elements to derive vector FmLARβV5GFP. Vector preparations made by co-transfection of 293T cells with vector and packaging plasmids had transducing titers from 1.8 x 107 to 3.0 x 107 TU/ml as assayed on HeLa cells. Three separate polyclonal 293T cell populations transduced 3 times at high vector concentration had copy numbers of the SIN-proviral genomes, as measured by RealTime PCR, that averaged 41 with a range of 23–68. Vector mobilization was evaluated by transfection of these populations with packaging plasmids. Conditioned media harvested from the transfected cells were then assayed for transfer of the GFP marker on HeLa cells. The mobilized titers were normalized based on vector copy number. The mobilized titer of the MSCV-U3 was 38,000 ± 3500 and that of d432βGFPim was only 810 ± 100 (p = 0.0004). The mobilized titers of the vectors in which the globin regulatory elements were in the forward orientation were also significantly lower than MSCV-U3; Fd432βGFP was 2100 ± 250 (p = 0.005) and FmLARβV5GFP was 1600 ± 120 (p = 0.0005). Those data suggest that the globin LCR elements are less likely than the retroviral LTR to induce transcription of the integrated vector genome in nonerythroid cells. Our results combined with ongoing analysis of the influence of vector integration on expression of surrounding genes in separate studies will provide a safety profile of globin lentiviral vectors to guide the development of future clinical protocols.

scholarly journals Reduced Mobilization of Rev-Responsive Element-Deficient Lentiviral Vectors

2005 ◽  
Vol 79 (14) ◽  
pp. 9359-9362 ◽  
Author(s):  
Susann Lucke ◽  
Thomas Grunwald ◽  
Klaus Überla
Keyword(s):  
Human Immunodeficiency Virus ◽  
Lentiviral Vector ◽  
Binding Protein ◽  
Lentiviral Vectors ◽  
Target Cells ◽  
Novel Approach ◽  
Immunodeficiency Virus ◽  
Efficient Vector ◽  
Responsive Element ◽  
Hiv 1

ABSTRACT Infection of cells transduced with a lentiviral vector by human immunodeficiency virus (HIV) could lead to packaging of the lentiviral vector RNA into HIV particles and unintended transfer of the vector. To prevent this, the Rev-responsive element (RRE) of an HIV-1 vector was functionally replaced by a heterologous RNA element (MS2). Providing Rev fused to an MS2 binding protein allowed efficient vector production. Mobilization of the vector from infected target cells was below the level of detection and at least 103- to 104-fold lower than for the RRE-containing vector. Thus, RRE-deficient lentiviral vectors provide a novel approach to reduce the risk of vector mobilization.

Establishing Safety in the Clinic for the First Lentiviral Vector To Be Tested in Humans.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 411-411
Author(s):  
Boro Dropulic ◽  
Laurent Humeau ◽  
Vladimir Slepushkin ◽  
Xiaobin Lu ◽  
Peter Manilla ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Opportunistic Infections ◽  
Lentiviral Vector ◽  
Lentiviral Vectors ◽  
Envelope Gene ◽  
Rev Response Element ◽  
Cd4 Counts ◽  
Viral Loads ◽  
Gene Therapy Application ◽  
Wide Range

Abstract Lentiviral vectors (LV) offer improved therapeutic benefit of gene therapy applications as a result of their ability to stably transduce a wide range of cycling and non-cycling cells. The first Phase I clinical trial using a lentiviral vector, VRX496, was initiated in January of 2003. The vector evaluated is an HIV-1 based lentiviral vector, fully gutted but retaining the 5′ and 3′ long terminal repeats, the packaging sequence, cPPT/CTS, splice donor and splice acceptor, and rev response element (RRE). VRX496 contains a splice-independent 937-base antisense sequence targeting the envelope gene in the HIV genome, and a small 186-base tag derived from the GFP gene to serve as a molecular marker for vector in HIV infected patient cells. Five (5) HIV-infected patients with CD4 counts between 200 and 500, and viral loads above 5000 copies/ml, no history of opportunistic infections, who have failed two regiments of highly active antiretroviral therapy (HAART), were serially enrolled in the trial. After leukopheresis, patient’s CD4 T lymphocytes were isolated by negative selection of CD8+ cells, and then cultured for three days in the presence of immobilized CD3/28 beads, and VRX496. Thereafter, the vector and beads were removed, cells were expanded to the dose of 1 x 1010, and then given intravenously over 15 minutes. At the time of this abstract, 4 patients have been dosed and have passed the initial 21-day safety assessment; a fifth patient is scheduled for dosing. No serious adverse events have occurred. All patients have steady CD4 counts, and viral loads have decreased below baseline in all patients, notably from 218,000 pre dose to 36,000 1 year post dosing in the first patient, and from 22,000 pre dose to 1,625 at 9 months post dosing in the second patient; patients 3 and 4 are at 3 months and 21 days post-infusion, respectively. Importantly, persistence of vector-modified CD4 cells are detected in the peripheral blood out to 9 months post dosing. This trial establishes for the first time the safety of lentiviral vectors for clinical gene therapy application.

scholarly journals Development of a forward-oriented therapeutic lentiviral vector for hemoglobin disorders

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Naoya Uchida ◽  
Matthew M. Hsieh ◽  
Lydia Raines ◽  
Juan J. Haro-Mora ◽  
Selami Demirci ◽  
...  
Keyword(s):  
Gene Therapy ◽  
High Efficiency ◽  
Lentiviral Vector ◽  
Transduction Efficiency ◽  
Specific Expression ◽  
Hematopoietic Stem ◽  
Rev Response Element ◽  
High Level ◽  
Intron 2

Abstract Hematopoietic stem cell (HSC) gene therapy is being evaluated for hemoglobin disorders including sickle cell disease (SCD). Therapeutic globin vectors have demanding requirements including high-efficiency transduction at the HSC level and high-level, erythroid-specific expression with long-term persistence. The requirement of intron 2 for high-level β-globin expression dictates a reverse-oriented globin-expression cassette to prevent its loss from RNA splicing. Current reverse-oriented globin vectors can drive phenotypic correction, but they are limited by low vector titers and low transduction efficiencies. Here we report a clinically relevant forward-oriented β-globin-expressing vector, which has sixfold higher vector titers and four to tenfold higher transduction efficiency for long-term hematopoietic repopulating cells in humanized mice and rhesus macaques. Insertion of Rev response element (RRE) allows intron 2 to be retained, and β-globin production is observed in transplanted macaques and human SCD CD34+ cells. These findings bring us closer to a widely applicable gene therapy for hemoglobin disorders.

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