Lantiviral Vectors Fazal Tabassam Ph.D Feb.16,2009

A Retrovirus is a ribonucleic acid (RNA) virus that must reverse to the DNA before reproducing / replicating. DNA gene that enables the Virus to replicate. HIV belongs to the subclass of lentiviruses or Slow viruses. Characterized by a long interval between infection and development of disease.

Retroviral vectors derived from oncoretroviruses such as the murine leukemia virus (MLV) Most widely used gene therapy tools for transgene delivery in the clinics. The attractiveness of oncoretroviral transduction technologies resides in: Ability to mediate stable integration in the target chromosomes likely promoting long-term expression of delivered transgenes.

(ii) Large cloning capacity sufficient for most foreseeable clinical situations; (iii) Compatibility with pseudotyping strategies which extend the tropism of gene delivery; exclusive delivery of therapeutic transgenes in the absence of viral genes which precludes any potent humoral immune response enables recurring treatments.

The use of retroviral vectors for delivery of genes has been well established in the last few years. Can accommodate long sequences, the products of which will be stably expressed due to integration into the cell chromosome. Non-immunogenic due to the lack of viral coding sequences transfer. Their properties make them very attractive for use in gene therapy.

Lentiviral Vectors Based on the human immunodeficiency virus type 1 (HIV-1) and related complex retroviruses. Capable of efficiently transducing nondividing and slowly dividing cells, including hematopoietic stem cells, resulting in stable integration and sustained transgene expression. Increasingly used for preclinical models of gene therapy and other forms of experimental transgenesis, including oncogene delivery.

Construction of the sin-HIV-1 based vector 2 3 1 # The picture shows all the proteins of HIV-1 are driven by the CMV promoter. For the actual production of virus, most of the proteins are deleted as in the second and third generation of HIV-1 based vectors. 2 # Due to the extremely low efficiency of pseudotyping HIV-1 with HIV-1 envelope in trans, envelopes of other viruses have been employed efficiently, such us GalV, VSV-G, and MLV. 3 # Different versions of the delivery vectors

HIV is a primate lentivirus Lentiviruses can infect nondividing cells Replication driven from long terminal repeats Structural genes - gag, pol, env Regulatory genes - tat, rev Accessory genes - vif, vpr, vpu, nef

Lentiviruses can replicate in non-mitotic cells due to pre-integration complex, a macromolecular structure comprising the viral genome, a few structural proteins and the enzymes responsible for reverse transcription and integration.

Vectors based on the lentiviruses subclass of Retroviridae have the special characteristic of being able to transduce non-dividing cells. But bearing in mind that the most characterized member of the lentiviruses that naturally infects human cells is HIV-1. So there remain safety concerns.

Lentiviral vectors Efficient tools for gene transfer into various dividing and non-dividing target cells. Offer several advantages over other vectors Lack of transfer of viral genes relatively large capacity for therapeutic genes. Demonstrated ability to achieve efficient and sustained transgene expression Recently been approved for human clinical studies.

Like all retroviruses, the HIV-1 genome contains the gag, pol and env regions, which encode the core proteins, the virion-associated enzymes and the envelope (Env) glycoprotein. This coding region is flanked by the long terminal repeats (LTRs; 5' and 3'LTR) and cis-acting sequences essential for integration, transcription and polyadenylation.

In addition, HIV-1 contains two regulatory genes, tat and rev, required for viral replication (LTR transactivation and nuclear export of viral RNA) and four accessory genes, vif, vpr, vpu and nef, which are dispensable for viral growth but critical for in vivo replication and pathogenesis.

Basal transcription of the HIV-1 provirus’ 5'LTR initially results in small amounts of multiply spliced transcripts encoding Tat, Rev and Nef. Tat transactivates 5'LTR-mediated transcription until Rev reaches a threshold concentration and mediates cytoplasmic accumulation of unspliced and singly spliced viral transcripts followed by production of the late viral proteins.

HIV-1 genome: Structure of a non-RCR-HIV-1 based vector

Sin vectors Although the recombination event possible between three independent plasmids is extremely small, ways to decrease even further this probability when dealing with HIV-1 based vectors have been undertaken in the plasmids presented at this site. This is the basis of the development of self-inactivating vectors or SIN vectors.

Sin vectors Basically, most of the U3 region of the 3’LTR is deleted. This site harbors the major transcriptional functions of the HIV genome. During the process of reverse transcription, the 3’LTR is copied to the 5’LTR. By deleting non-replicative portions of the 3'LTR, the genomic viral DNA is inserted into the target genome as a promoter-less sequence. The lack of active viral promoter avoids both the possible transcription of the viral sequence and detrimental effects on eukaryotic gene expression.

Viral Particles Usually, particles are generated using the safety-enhanced ‘‘third-generation’’ split plasmid system that consists of a plasmid encoding the Rev-dependent genomic vector RNA with self-inactivating long terminal repeats (LTRs), two plasmids expressing the HIV-1 proteins Rev and Gag-pol, and a fourth plasmid encoding the envelope protein of choice.

A variety of viral and non-viral vectors and expression concepts have been designed and evaluated for their safety, high-level transduction, tropism and sustained expression in a variety of therapeutically relevant cells and tissues

RNA interference( RNAi) Process in which small dsRNA, known as siRNA (small interference RNA) target sequence-specific RNAs and trigger specific gene silencing Gene silencing allows us to study gene function validate function of genes hopefully discover various new drugs, therapeutics, and develop treatments such as for cancer, AIDS, and others.

Lentiviral shRNA Advantages Gene- and sequence-specific high level of knockdown long-term gene silencing can be transduced into both dividing and nondividing cells as well as primary cells stable gene suppression identifying genes is fast can be used in vivo and in vitro

primary cells, stem cells, and some tumor cell lines are resistant to the transfection methods transfection is short-term as transfected cells are dividing, [ siRNA] is diluted RNA increases and is expressed (lentiviral) shRNA can overcome these problems !