Tel: 1-631-626-9181 (USA)   44-207-097-1828 (Europe)
  Email:

Lentiviral Guide


CBpromise   

Our promise to you:
Guaranteed product quality, expert customer support.

24x7 CUSTOMER SERVICE
CONTACT US TO ORDER

Lentiviral Guide

Introduction and Background

Lentiviruses comprise a genus of the Retroviridae family. All Retroviruses share similarities in structure, genomic organization and replicative properties. Retroviruses are spherical viruses of about 80-120 nm in diameter and are characterized by a genome comprising two positive-sense single-stranded RNA. In addition, they have a unique replicative strategy where the viral RNA is reverse transcribed into double-stranded DNA that is integrated into the cellular genome. Together with the RNA strands, the enzymes necessary for replication and the structural proteins form the nucleocapsid. The latter is inside a proteic capsid which is surrounded by a double lipidic membrane. Connecting the lipidic membrane and the capsid there are the matrix proteins. The lipidic membrane has its origin in the host cells and presents at the surface the envelope viral glycoproteins (Env) (Figure 1).

Lentiviral Guide Figure 1. Schematic structure of a retroviral particle.

Lentiviral Expression Systems

Lentiviral expression vectors are the most effective vehicles for transducing and stably expressing different effector molecules (cDNA, siRNA, DNA fragments, antisense, ribozymes, etc.) or reporter constructs in almost any mammalian cell, including nondividing cells and whole model organisms. As with standard plasmid vectors, it is possible to introduce lentiviral constructs in plasmid form into the cells with low-to-medium efficiency and get transient expression of effectors (reporters) using conventional transfection protocols. By packaging the lentiviral expression construct into pseudoviral particles, you can obtain highly efficient transduction, even with the most difficult to transfect cells, such as primary, stem, and differentiated cells.

The expression construct transduced in cells is integrated into genomic DNA and provides stable, long-term expression of cDNA, siRNA or reporter gene. Endogenously expressed siRNA effectors provide long-term silencing of the target gene and allow the researcher to generate cell lines and transgenic organisms with a stable knockdown phenotype for functional studies. Expression of full-length cDNAs from integrated viral constructs is a unique tool to study gain-of-function effect for cellular phenotypes. Stably integrated transcriptional reporter constructs are a novel approach to the study of transcriptional regulation in the natural chromosomal environment and the monitoring of specific signaling pathways. In addition, lentiviral delivery does not produce the nonspecific cell responses typically associated with chemical transfections or use of an adenoviral delivery system.

Lentiviral Components

In order to increase the safety of lentivirus, the components necessary for virus production are split across multiple plasmids. Four generations of lentiviruses are currently considered; these differ from each other in terms of the number of genetic constructs used to drive the expression of the viral components, the number of wild-type genes retained as well as the number and type of heterologous cis-elements used to increase vector titers and promote vector safety. While a number of lentiviral vector systems are based on transduction of two helper plasmids (second generation) with the transfer plasmid, some newer systems (third generation) have the packaging and envelope constructs on three plasmids which are combined with the transfer plasmid. The typical transfer and two helper plasmids used in replication-defective retroviral or lentiviral production are:

  • Transfer Vector

This vector is used to transfer genes of interest into the target cells. There is a deletion of the U3 region and other transcriptionally active sequences from the 3’ LTR, which leads to it being a self-inactivating LTR. The 5’ LTR drives expression of the packaged genomic RNA, and the transgene is driven from a promoter within the vector.

  • Viral Enzymatic Proteins

Gag and Pol: These viral proteins are needed for maturation of the virion. Tat and Rev upregulate transcriptional activity and nuclear export of genomic RNA. The accessory genes have been deleted to increase safety by reducing the probability of recombination. Tat has been removed in newer generations of lentiviral systems and Rev has been placed on a separate vector to increase safety.

  • Envelope Protein

Viral vectors can be pseudotyped with coat proteins from other pathogens to alter their tropism. The most commonly used is a fusogenic envelope G glycoprotein of the vesicular stomatitis virus (VSV-G). Other common envelope proteins are derived from rabies virus, Ebola, MLV, baculovirus, filovirus and measles virus.

Lentiviral Guide Figure 2. Schematic representation of the four generations of lentiviral packaging constructs: A) First generation packaging vector. B) Second generation packaging vector. C) Third generation packaging vector. D) Fourth generation packaging vector.

Transient Lentiviral Vector Production

Commonly lentiviruses are produced by co-transfecting cells with the several expression cassettes harboring the transgene and the viral elements using chemical agents (such as polyethylenimine or calcium phosphate) and after 24 to 72 hours the lentiviruses are harvested. This production system is fast and can be easily adapted to produce lentiviruses with new genes of interest or with other Env glycoproteins. It is a simple process to apply at small scales commonly used in research, unlike the laborious development of a packaging cell line. However transient production is not the ideal choice for large and clinical lentiviruses productions as it is difficult to scale-up and requires large amounts of Good Manufacturing Practices (GMP) grade plasmid expressions cassettes turning the production more expensive. Moreover, transient LV production brings some biosafety problems like recombination between expression cassettes which could originate or facilitate the RCL formation.

Stable Lentiviral Vector Production

To overcome the biosafety problems in lentivirus transient productions, inducible packaging cells lines have been developed. The development of these systems is more time-consuming because after insertion of each expression cassette the population of stably transfected cells is usually screened for the best producer clone, like for γ-RVs, to maximize the lentivirus production. However, these packaging cell lines are derived from one clone. Thus, all the cells have the same growth and lentivirus production behavior being the lentivirus productions reproducible. This allows the generation of GMP cell banks, increasing safety conditions. In conditional packaging cell lines the expression of cytotoxic proteins is under control of inducible promoters and the number of cells and growth conditions can be controlled, starting the LV production at a defined moment via adding an inductor or removing the suppressor from the culture medium. Originally the titers were low but further improvements in the expression cassettes design and optimization of the induction parameters resulted to similar levels of transient productions.

Lentiviral-Mediated Gene Therapies

Third-generation lentiviral vectors represent some of the safest and easiest-to-use vectors available for the delivery of genes into mammalian tissue. The advantages of these engineered viral vector-based gene therapies are that they effectively deliver genetic material and maintain long-term stable expression in target cells, deliver larger amounts of genetic material than other manners, are nonpathogenic, and do not cause an inflammatory response in the recipient. Therefore, this system is increasingly the vector of choice for in vivo and ex vivo delivery of genes to be used in gene and cell therapies, respectively.

Although lentiviral vectors are derived from human immunodeficiency virus type 1 (HIV-1) and related wild-type viruses, modified lentiviral vectors are nonpathogenic and not capable of replication after the initial gene delivery event. The RNA-based vector has been engineered to deliver genes to cells and tissues safely and effectively for the purposes of gene therapy. Specifically, lentiviruses can deliver up to 8.5 kB of genetic material, which is then reverse transcribed in the target cell and integrated into the genome to achieve long-term stable expression. The use of lentiviruses for gene therapy includes HIV-based vectors and non-HIV-based vectors. Over the past several years, clinical trials in Europe and the United States have shown safety and initial indications of efficacy for various lentivirus-mediated gene therapies.

References:

  1. Tomás H A, et al. Lentiviral gene therapy vectors: challenges and future directions. Gene Therapy-Tools and Potential Applications. InTech, 2013.
  2. White M, et al. A Guide to Approaching Regulatory Considerations for Lentiviral-Mediated Gene Therapies. Human Gene Therapy Methods, 2017, 28(4):163-176.
  3. Singer O, Verma I. Applications of lentiviral vectors for shRNA delivery and transgenesis. Curr Gene Ther. 2008;8:483-8
  4. Lemiale F, Korokhov N. Lentiviral vectors for HIV disease prevention and treatment. Vaccine. 2009;27:3443-9

Quick Inquiry

   Please input "biogene" as verification code.