What type of nucleotide is packaged inside a lentivirus

HIV integration site selection: targeting in macrophages and the effects of different routes of viral entry. Mol Ther ; 14 : — Mol Ther ; 13 : — Lentiviral vector integration profiles differ in rodent postmitotic tissues.

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Cell Ther Transplant ; 1 : 84— Download references. You can also search for this author in PubMed Google Scholar. Correspondence to R Daniel. Reprints and Permissions. Papayannakos, C. Understanding lentiviral vector chromatin targeting: working to reduce insertional mutagenic potential for gene therapy. Gene Ther 20, — Download citation. Received : 11 May Revised : 04 September Accepted : 05 September Published : 22 November Issue Date : June Anyone you share the following link with will be able to read this content:.

Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. Cardiovascular Drugs and Therapy Molecular Neurodegeneration Scientific Reports Advanced search. Skip to main content Thank you for visiting nature. Download PDF. Subjects Gene delivery Gene therapy Viral vectors. Abstract Replication-deficient retroviruses have been successfully utilized as vectors, offering an efficient, stable method of therapeutic gene delivery.

Introduction Engineering retroviruses into vectors of therapeutic value has been a highly pursued effort. LVs display an effective mode of gene transfer in pre-clinical experiments Vector engineers have successfully exploited the integrative property of lentiviruses to incorporate nucleic acid into the chromosomes of non-replicating or terminally differentiated cells. LVs have been clinically tested demonstrating safe and successful outcomes It should not go without stating that LV are not the only systems that have made it to clinical testing.

Differentiating these from natural HIV-1 infection will, therefore, be important, and require more extensive testing. Beyond the ex vivo modification of cells for adoptive transfer back into the patient, lentiviral vectors are also being applied directly in vivo for therapeutic purposes. In vivo gene delivery using a lentiviral vector has also been applied clinically to the eye [ 80 ].

These approaches face a number of hurdles including efficiency, need for tissue-restricted promoters, and immunogenicity. The latter is particularly important since immunogenicity can be related to both the delivered gene as well as components of the vector. As discussed earlier, the lentiviral vector envelope captures membrane proteins from the packaging cell lines during the budding process.

Alloimmune reactivity towards HLA class I proteins carried within the vector envelope have been described and can limit vector survival. Gene editing of packaging cells to generate lines that lack HLA class I can enhance the stability of lentiviral vectors in serum [ 81 ]. Surface engineering in addition to vector genome engineering will likely be critical for successful application of these vectors in vivo. The third-generation SIN lentiviral vector system makes the generation of RCLs very unlikely because the viral genome is split into separate plasmids.

The requirements for follow-up vary depending on the country; in the United States, monitoring of patients at least once a year for 15 years after receiving gene therapy is recommended. The long-term post-marketing surveillance required for the tisagenlecleucel CTL; Kymriah that was recently approved by the FDA was in part driven by these theoretical concerns [ 85 ]. However, the optimal duration of patient follow-up depends on several factors, including vector persistence and transgene expression.

The FDA also requires that patients participating in clinical trials that use viral vectors receive information about their mechanism of action and the possible effects of DNA integration, including the risk of delayed malignancies [ 86 ]. To help establish follow-up procedures and collect data from patients treated with gene therapies, clinical trials for the follow-up of patients treated with specific gene therapy products are ongoing.

Several ongoing areas of research are aimed at further improving gene therapy with novel viral vector designs. Non-integrating lentiviral vectors NILVs have been investigated as a means of avoiding insertional mutagenesis. NILVs, which are deficient in the viral integrase protein, can transduce both dividing and non-dividing cells, and the viral genome remains present in the cell as an episome rather than integrating into the host genomic DNA [ 87 ].

It is expected from the non-integrating genome structure of NILVs that gene expression might be short-lived in dividing cells. Although this may be undesirable in some applications, this dilutional effect might be useful in some settings, such as CAR T cells, where long-term expression of the genetic payload may not be necessary. NILVs have been used effectively as a vaccination strategy in pre-clinical models, resulting in cellular and humoral immunity as well as anti-tumor immunity [ 88 ].

NILVs can also be co-transduced into cells with zinc finger nucleases, which facilitate recombination of DNA encoded in the vector with a specific site in the host DNA [ 89 ]. Replacing the endogenous TCR in this manner results in abrogation of off-target activity mediated by the endogenous TCR, thereby enhancing safety. To allow for long-term expression in dividing cells, a dual NILV vector system was developed to include the integrase of phage phiC31 [ 91 ].

Two types of cancer vaccines using lentiviral vectors have been investigated: dendritic cell vaccines and cancer cell vaccines. Dendritic cells loaded with peptide from a tumor antigen can be used as a vaccine against cancers expressing that antigen. One such dendritic cell vaccine, Sipuleucel-T, has been approved by the FDA as a prostate cancer therapy. Lentiviral vectors have been investigated as a method of expressing tumor antigens in or modifying co-stimulatory signals on dendritic cells to further enhance their efficacy [ 92 , 93 ].

Lentiviral vectors have also been used to constitutively activate the MAP kinase pathway in dendritic cells, which resulted in enhanced anti-tumor responses in mice [ 94 ]. An alternative vaccine approach is the use of cancer cells, which already express the tumor antigens of interest, instead of dendritic cells, which must be loaded with peptide.

A study of B-cell lymphoma cells lentivirally transduced with co-stimulatory proteins and interleukin demonstrated that using these modified cells as a vaccine can result in enhanced immunogenicity to the parental lymphoma cell lines in murine models [ 95 ]. Lentiviral vectors have also been used to convert the K erythroleukemic cell line into artificial antigen-presenting cells that can be used for in vitro T-cell expansion and potentially in vivo vaccination similar to the previously-reported GVAX [ 96 , 97 ].

Although more research is needed to determine clinical efficacy and safety of cancer vaccines developed using lentiviral vectors, these approaches may lead to novel therapeutic options for patients. The CRISPR-Cas9 system uses an RNA sequence to guide the Cas9 nuclease to create precise double-strand breaks, which can then be followed by homologous recombination to result in gene deletion or point mutations [ 98 ].

One study used a lentiviral CRISPR guide RNA library to introduce targeted mutations into embryonic stem cells, and subsequent identification of phenotypic mutants demonstrated the efficacy of this approach as an alternative to genetic screening using RNA interference [ 99 ].

There is some concern around the amplification of off-target effects with gene editing as a result of integrating viruses due to the persistent expression of the gene editing machinery. In order to overcome these potential limitations, Chen et al.

Short-term delivery via NILVs might also circumvent some of these challenges. Lentiviral vectors are also frequently used in the research setting to alter gene expression through the expression of short hairpin RNA or antisense RNA.

Most of these approaches are still in early stages of development, and much further research is needed to determine whether lentiviral vectors can serve as a viable platform for delivering these gene editing tools for therapeutic purposes.

Gene therapy using lentiviral vectors has emerged as a promising therapeutic option for several conditions. The first lentivirally transduced cellular therapy, tisagenlecleucel CTL, Kymriah , was approved in the United States in August of for the treatment of pediatric and young adult patients with acute lymphoblastic leukemia. Several additional cellular therapies based upon lentiviral vector-engineered cells are in late-phase development. Third-generation SIN lentiviral vectors, in particular, have demonstrated safety when used to transfer genes into both stem cells and T cells.

Although there is a theoretical potential for insertional oncogenesis with lentiviral vectors, no cases have been reported with a natural HIV or gene therapy using lentiviral vectors. Continued follow-up of patients who have already received lentiviral vector-based gene therapies is still necessary to understand the long-term safety and efficacy of these vectors.

Additional basic and clinical research to improve transduction efficiency and manufacturing are also still needed. Maude SL. Future directions in chimeric antigen receptor T cell therapy. Curr Opin Pediatr. Progress and problems with the use of viral vectors for gene therapy. Nat Rev Genet. Escors D, Breckpot K. Lentiviral vectors in gene therapy: their current status and future potential. Arch Immunol Ther Exp Warsz. Craigie R, Bushman FD. Host factors in retroviral integration and the selection of integration target sites.

Microbiol Spectr. Meng B, Lever AM. Wrapping up the bad news: HIV assembly and release. Association of integrase, matrix, and reverse transcriptase antigens of human immunodeficiency virus type 1 with viral nucleic acids following acute infection. Nat Rev Microbiol. The Vpr protein of human immunodeficiency virus type 1 influences nuclear localization of viral nucleic acids in nondividing host cells. Complementary assays reveal a relationship between HIV-1 uncoating and reverse transcription.

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Genome Res. Yamashita M, Emerman M. Capsid is a dominant determinant of retrovirus infectivity in nondividing cells. Impact of nucleoporin-mediated chromatin localization and nuclear architecture on HIV integration site selection. Nuclear architecture dictates HIV-1 integration site selection. Role of T cell activation and expression of the T4 antigen. J Immunol. Bukrinsky M, Stanwick T. Still, the image data collected from thin slices can be assembled into 3-D images using computational algorithms.

The titre of a lentiviral vector depend to a large degree on the type of the marker used for analysis. Indeed, the titre determined by detecting the fluorescent transduction markers is usually higher than the titre obtained by counting drug resistant cell clones. Genes for luminescence proteins can be transferred by lentiviral vectors and used as cell tracers Reumers, Deroose et al.

Luciferases from North American firefly Photinus pyralis and sea pansy Renilla reniformis are commonly used. A version of firefly luciferase with enhanced expression in mammalian cells luc2 was developed by Promega by codon optimization as discussed in Section 2. PEST is a 40 amino acid sequence present in the C-terminal region of mouse ornithine decarboxylase.

CL1 is a 16 amino acid degron from yeast. This signal was also optimized for expression in mammalian cells. Versions of luciferase with the degradation signals improve responsiveness to factors enhancing or inhibiting luciferase expression. Other specialized versions of luciferases are available. For example, secreted firefly luciferase is convenient to measure the luciferase activity of live cells in tissue culture.

Renilla luciferase-neo R fusion protein was generated and can be used for both cell clone selection and as an internal luminescence control. However, low spatial resolution limits due to light dispersion in the animal restrict the expediency of this approach. Surface antigen markers, e. The intracellular domains of these proteins are removed to avoid signal transduction and the extracellular domains are supplied with GPI-lipidation signal for plasma-membrane anchoring as in Miltenyi Biotec MAC Select TM system.

Background signal during whole-animal imaging can be avoided by using species-specific monoclonal antibodies for the cell surface marker proteins in a heterologous host organism. In general, cis -acting elements strictly required for gene expression in eukaryotes are a minimal promoter and a transcription terminator.

Genes coding for proteins cistrons also necessarily contain: 1 the translation start codon ATG with the surrounding Kozak consensus sequence controlling translation initiation; 2 the protein coding sequence; 3 the stop codon. The Kozak consensus sequence is important for expression of protein-coding transgenes born on lentiviral vectors.

The Polymerase II transcripts, e. Polyadenylation of eukaryotic mRNAs is important for their protection from exonucleolytic attack and for their export out of nucleus. It also serves as a means of transcription termination. For mRNA to be polyadenylated, it should contain a specific signal sequence downstream of its polypeptide coding sequence. Thus, foreign pA signals within lentiviral vectors should be either avoided altogether, or weakened, or positioned to terminate the transcription of the anti-genomic DNA strand only.

With this challenge in view, it should be noted that while most pA signal sites act unidirectionally, a pA signal borrowed from SV40 viral genome is known to terminate RNA and to promote polyadenylation irrespective of transcription direction. A brief look at the transcription map of SV40 can explain this fluke. Indeed, two opposing transcription waves of SV40 meet and terminate at its polyadenylation signal site, which is, therefore, an overlap of two opposing polyadenylation signals.

Size limitations of the lentiviral payload and insufficiently precise enhancer localization data restrict the use of enhancers in the lentiviral vectors. However, small enhancer elements can still be used where, for example, tissue-specific or inducible transgene expression is desired.

A typical eukaryotic protein-coding gene is a patchwork of coding exons and non-coding introns, so that the translation-grade mRNA is produced by the splicing of the primary transcript. As the payload space within the lentiviral vectors is limited, the standard practice is to include the genes in their complementary DNA cDNA form, that is, as spliced versions without introns.

The introns, however, are not entirely inert genetically and occasionally take part in the regulation of gene expression. In such situations, the inclusion of small regulatory introns within lentiviral vectors can be considered Le Hir, Nott et al. Coding sequences delivered by lentiviral vectors are often derived from non-mammalian organisms where the translation machinery is adapted to a non-mammalian profile of codon frequencies. Therefore, the optimization of codon frequencies for the genes, which are born on the lentiviral vectors, is often advantageous.

The stability of genomic RNA of lentiviral vectors is crucial for attaining high lentiviral vector titres and stability of the lentiviral vector encoded mRNAs is important for efficient transgene expression. It was discovered that an element from Woodchuck Hepatitis Virus WHV genome can operate at a post-transcriptional level to improve transgene expression.

Cistrons are not the only cargo genes delivered by the lentiviral vectors. The benefits of the lentiviral vectors, such as the relative stability of transgene expression and the ability to transduce postmitotic cells, considerably broaden the versatility of gene knock-down experiments with shRNAs Rubinson, Dillon et al.

In summary, cis -acting elements regulating gene expression act in concert and in a cell-type-specific manner. The inherent mosaicism of lentiviral genetic organization allows interchangeable use of an extensive array of genetic elements for the generation of new lentiviral vectors. However, some combinations of genetic determinants are less functional than others, so the optimization of the lentiviral vector set-up is usually required. It is a relatively common occurrence for transgene expression to die out both in terms of the reduction of the fraction of expressing cells and the decrease of the efficiency of expression.

Integrated lentiviral proviruses are faithfully maintained in mammalian cells, so the reasons for the shutdown of transgene expression are mostly epigenetic. Certainly, different promoters have various capabilities to maintain long-term transgene expression.

In particular, some promoters tend to turn off in cell populations where they are not normally active. The shutdown of transgene expression is particularly common in cell populations undergoing differentiation Bagchi, Kumar et al.

Natural chromosomal integration of lentiviruses tends to occur in transcriptionally active areas of the genome where heterochromatin and DNA methylation are unlikely to interfere with transgene expression. However, as the cells differentiate, the pattern of heterochromatization and DNA methylation changes and some of the proviruses find themselves in the transcriptionally silent areas of the genome.

There are many levels at which the longevity of transgene expression can be addressed through the lentiviral vector design, including: 1 control of the provirus amenity to methylation e. In principle, the protection of proviruses from heterochromatin can be achieved with genomic insulators or other similar anti-heterochromatin elements.

However, experiments with known insulators show that their effects on transgene expression from lentiviral proviruses are multi-vectorial depending on the cell context Grandchamp, Henriot et al. Ideally, targeting proviruses to a continuously active locus e. An alternative solution is to escape chromatin-remodeling events by creating episomally maintained lentiviral proviruses Section 5. The fairly large size of the lentiviral vector backbone plasmids means they contain a limited number of unique sites for restriction nucleases.

Thus, it is often desirable to introduce artificial clusters of suitable unique restriction sites polylinkers to simplify the modification of these plasmids. Alternatively, selection schemes involving site-specific recombination can be used for repetitive modifications, e. In this scenario, the introduction of suitable site-specific recombination sites into the lentiviral vector backbone plasmid is required. As discussed in Section 2. These plasmids are particularly vulnerable during initial establishment in bacteria.

Instability of nascent recombinant plasmids can result in a practical unfeasibility of seemingly straightforward DNA cloning strategies. For example, the generation of new lentiviral vector backbone plasmids through inefficient ligation of two DNA fragments with blunt ends is normally very challenging.

In such situations we recommend splitting the DNA cloning procedure into separate cloning steps, each one relying on either effective positive selection of new recombinant plasmids or on efficient ligation. In this approach, a plasmid containing the desired insert and marked with antibiotic resistance marker 1 is first fused with the lentiviral vector backbone plasmid marked with antibiotic resistance marker 2 using positive selection of the co-integrate plasmid with two antibiotics.

The desired new lentiviral vector backbone plasmid is then obtained by removing the unwanted plasmid sequences from the resultant bi-replicon plasmid using restriction digestion and efficient intra-molecular ligation reaction.

The genome sizes of non-defective wild type HIV-1 isolates are close to 9. The lentiviral packaging size constraints are dictated by the geometry of the viral capsid and are thought to be fairly permissive of the smaller than wild type genome versions, but remarkably intolerant of the larger than wild type variants.

As lentiviral sequences required for genomic RNA packaging and chromosomal integration constitute about 2 kb, the available gene payload space within HIV-1 based lentiviral vectors should not be much more than 7.

Thus, the insert size capacity of the lentiviral vectors is completely appropriate for the vast majority of the monogenic applications but can present a challenge in situations where the delivery of several genes by a single vector is required. Therefore, various possible methods of gene cargo reduction have been explored. The expression of two or more cistrons from a single promoter can be achieved by the employment of internal ribosome entry site IRES elements, which are normally borrowed from viral genomes Fux, Langer et al.

Most lentiviral systems use a transfer plasmid and two helper plasmids. Their features are described in detail in the text. Newer systems may employ three helper plasmids with the rev gene being encoded on a separate plasmid.

This is believed to improve safety by further preventing recombination events from producing replication-competent virus. Coli cells SV40 Origin Provides for stable propagation of the plasmid in packaging cells F1 Ori origin of replication. Enhancement of lentivirus-based transduction and improvement of the functions of the transduced cells.

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