Lentivirus Gene Expression Vector (KanR)

Overview

Replacing an ampicillin resistance marker with a kanamycin resistance marker is one of the simplest ways researchers can ensure their vectors are translatable to the clinic. Many regulatory agencies, including the United States Food and Drug Administration (FDA) and the European Medicines Agency (EMA), strongly discourage the use of ampicillin in the production of therapeutic agents. Kanamycin is therefore the superior option when designing vectors with translational aims.

VectorBuilder offers a dedicated consultation service to help translational researchers plan for and manage the transition from basic research, to preclinical studies, to clinical trials, to GMP-compliant production and commercialisation. For researchers intending to develop novel therapies, CliniVec is an essential tool to facilitate success from the first step.

The lentiviral vector system is a highly efficient vehicle for introducing genes permanently into mammalian cells. Presently, it is one of the two most commonly used methods for gene delivery into mammalian cells (the other being conventional plasmid transfection). Features that make this system so popular include the ability to choose which promoter will drive the gene of interest and the ability to infect a very wide range of cell types.

Lentiviral vectors are derived from HIV, which is a member of the retrovirus family. Wildtype lentivirus has a plus-strand linear RNA genome.

A lentiviral vector is first constructed as a plasmid in E. coli. It is then transfected into packaging cells along with several helper plasmids. Inside the packaging cells, vector DNA located between the two long terminal repeats (LTRs) is transcribed into RNA, and viral proteins expressed by the helper plasmids further package the RNA into virus. Live virus is then released into the supernatant, which can be used to infect target cells directly or after concentration.

When the virus is added to target cells, the RNA cargo is shuttled into cells where it is reverse transcribed into DNA and randomly integrated into the host genome. Any gene(s) that were placed in-between the two LTRs during vector cloning are permanently inserted into host DNA alongside the rest of the viral genome.

By design, lentiviral vectors lack the genes required for viral packaging and transduction (these genes are instead carried by helper plasmids used during virus packaging). As a result, virus produced from lentiviral vectors has the important safety feature of being replication incompetent (meaning that they can transduce target cells but cannot replicate in them).

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For further information about this vector system, please refer to the papers below.

Highlights

Our vector is derived from the third-generation lentiviral vector system. This system has been engineered to significantly reduce the risk of producing replication-competent virus, which is essential for clinical applications. Additionally, our vector It is optimized for high copy number replication in E. coli, high-titer packaging of live virus, efficient viral transduction of a wide range of cells, efficient vector integration into the host genome, and high-level transgene expression.

Advantages

Permanent integration of vector DNA: Conventional transfection results in almost entirely transient delivery of DNA into host cells due to the loss of DNA over time. This problem is especially prominent in rapidly dividing cells. In contrast, lentiviral transduction can deliver genes permanently into host cells due to the integration of the viral vector into the host genome. This is essential for stable cell engineering for cell-based therapies; for example, lentivirus transduction is commonly used to generate T cells that permanently express chimeric antigen receptors (CARs) for CAR-T cancer therapies.

High viral titer: Our lentiviral vector can be packaged into high titer virus. When lentivirus is obtained through our virus packaging service, the titer can reach >10⁸ transducing unit per ml (TU/ml). At this titer, transduction efficiency for cultured mammalian cells can approach 100% when an adequate amount of viral is used. At the commercialization stage of clinical products, high titre is essential for cost-effective production at scale.

Very broad tropism: Our packaging system adds the VSV-G envelope protein to the viral surface. This protein has broad tropism. As a result, cells from all commonly used mammalian species (and even some non-mammalian species) can be transduced. Furthermore, almost any mammalian cell type can be transduced (e.g. dividing cells and non-dividing cells, primary cells and established cell lines, stem cells and differentiated cells, adherent cells and non-adherent cells). Neurons, which are often impervious to conventional transfection, can be readily transduced by our lentiviral vector. Lentiviral vectors packaged with our system have broader tropism than adenoviral vectors (which have low transduction efficiency for some cell types) or MMLV retroviral vectors (which have difficulty transducing non-dividing cells).

Customizable internal promoter: Our vector is designed to self-inactivate the promoter activity in its 5' LTR upon integration into the genome. As a result, users can put in their own promoter to drive their gene of interest within the vector. This is a distinct advantage over our MMLV retrovirus vectors, which rely on the promoter function of 5' LTR to drive the ubiquitous expression of the gene of interest.

Relative uniformity of gene delivery: Generally, viral transduction can deliver vectors into cells in a relatively uniform manner. In contrast, conventional transfection of plasmid vectors can be highly non-uniform, with some cells receiving a lot of copies while other cells receive few copies or none.

Effectiveness in vitro and in vivo: A crucial stage of translational research is the  transduction of cells in live animals for preclinical studies. The step from animal models to clinical trials is often where therapeutic agents falter in regulatory compliance; therefore, antibiotic marker selection is a critical consideration even at preclinical stages.

Low risk of replication competence: The safety of our vector is ensured by two features. One is the partition of genes required for viral packaging and transduction into several helper plasmids; the other is self-inactivation of the promoter activity in the 5' LTR upon vector integration. As a result, it is essentially impossible for replication competent virus to emerge during packaging and transduction. Therefore, the risk both to researchers handling virus in production, and to patients receiving the therapeutic product, is minimal.

Compliance with regulatory standards: Many individuals have hypersensitivity to beta lactam, and small amounts of beta lactam are extremely difficult to detect in standard quality control. Therefore, regulatory agencies strongly discourage the use of beta lactam-based antibiotics such as ampicillin at any stage during the production of clinic-bound products. Kanamycin offers an effective, low-cost alternative to ampicillin.

Disadvantages

Limited cargo space: The wildtype lentiviral genome is ~9.2 kb. In our vector, the components necessary for viral packaging and transduction occupy ~2.8 kb, which leaves ~6.4 kb to accommodate the user's DNA of interest. When the vector goes beyond this size limit, viral titer can be severely reduced. Our vector is routinely used for inserting several functional elements besides the ORF of the gene of interest, such as a promoter and drug resistance. A large ORF plus these additional elements could exceed 6.4 kb, and the result could be compromised viral production.

Technical complexity: The use of lentiviral vectors requires the production of live virus in packaging cells followed by the measurement of viral titer. These procedures are technically demanding and time consuming relative to conventional plasmid transfection.

Key components

CMV promoter: Human cytomegalovirus immediate early enhancer/promoter. It drives transcription of viral RNA in packaging cells. This RNA is then packaged into live virus.

5' LTR-ΔU3: A deleted version of the HIV-1 5' long terminal repeat. In wildtype lentivirus, 5' LTR and 3' LTR are essentially identical in sequence. They reside on two ends of the viral genome and point in the same direction. Upon viral integration, the 3' LTR sequence is copied onto the 5' LTR. The LTRs carry both promoter and polyadenylation function, such that in wildtype virus, the 5' LTR acts as a promoter to drive the transcription of the viral genome, while the 3' LTR acts as a polyadenylation signal to terminate the upstream transcript. On our vector, 5' LTR-ΔU3 is deleted for a region that is required for the LTR's promoter activity normally facilitated by the viral transcription factor Tat. This does not affect the production of viral RNA during packaging because the promoter function is supplemented by the CMV promoter engineered upstream of Δ5' LTR.

Ψ: HIV-1 packaging signal required for the packaging of viral RNA into virus.

RRE: HIV-1 Rev response element. It allows the nuclear export of viral RNA by the viral Rev protein during viral packaging.

cPPT: HIV-1 Central polypurine tract. It creates a "DNA flap" that increases nuclear importation of the viral genome during target cell infection. This improves vector integration into the host genome, resulting in higher transduction efficiency.

Promoter: The promoter driving your gene of interest is placed here.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of your gene of interest is placed here.

WPREmut6: Woodchuck hepatitis virus posttranscriptional regulatory element containing six point mutations. It retains WPRE activity while preventing the production of the truncated WHV X protein by disrupting its putative promoter and translation start site.

3' LTR-ΔU3: A truncated version of the HIV-1 3' long terminal repeat that deletes the U3 region. This leads to the self-inactivation of the promoter activity of the 5' LTR upon viral vector integration into the host genome (due to the fact that 3' LTR is copied onto 5' LTR during viral integration). The polyadenylation signal contained in 3' LTR-ΔU3 serves to terminate all upstream transcripts produced both during viral packaging and after viral integration into the host genome.

SV40 early pA: Simian virus 40 early polyadenylation signal. It further facilitates transcriptional termination after the 3' LTR during viral RNA transcription during packaging. This elevates the level of functional viral RNA in packaging cells, thus improving viral titer.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Kanamycin: Kanamycin resistance gene. It allows the plasmid to be maintained by kanamycin selection in E. coli. Kanamycin is considered superior to ampicillin for vectors used in translational research.