Viral vectors in gene technology

Dmitri Chilov

Gene transfer
Strategies Transgene KO (knock-out) KI (knock-in) KD (knock-down) Gene (cDNA) Promoter Homologous recombination Homologous recombination siRNA Gene transfer methods Over expression, expression of dominant-negative mutant

DNA transfection Used in Basic biology Study, study and study!

DNA injections

Transduction (viral vectors) The goal in gene therapy is: replace a defective function, introduce additional function (as in cancer), prevent (as in a vaccine) disease

Gene therapy

Viral vector production

Transfect cells (packaging cells) with DNA vectors encoding viral particles Collect the medium (with viral particles) or lyse the cells Purify and/or concentrate virus prep Determine amount of viral particles and MOI (multiplicity of infection) Apply virus to target cells (in vitro) and tissues (in vivo)

Strategy for engineering a virus into a viral vector

Infection versus transduction Cis and Trans acting elements Split construct design Make it replication defective (self-inactivating viruses) MOI multiplicity of infections (transduction) does not necessarily correspond to number of virus particles

From Kay Naldini 2001

Transduction of a target cell

Genome integration versus Episomal expression: Pluses and minuses Integration Long-term expression but: Transgene silencing reported And activation of neighboring genes Episomal expression: Short term but in slow dividing Cells can last for long time

From Kay Naldini 2001

From virus to viral vector

Viruses are to be modified to become gene delivery tools:

Eliminate trans acting elements Essential trans acting elements to be identified and provided by a helper vector Modify cis acting elements (creating self-inactivating viruses) Modify transgene cassette varying promoters to achieve high and/or specific gene expression Pseudotyping process of producing viruses or viral vectors in combination with foreign viral envelope proteins to improve or broaden tropism, increase stability, reduce toxicity and immune response Modifying envelope proteins to improve or broaden tropism increase stability, reduce toxicity and immune response

VIRAL GENE DELIVERY FOR TREATMENT OF HUMAN DISEASES

Best viral vector will have:

- No toxicity or immune response - long lasting transgene expression (with a rapid onset) - large packageing capacity (to include promoter, transgene, regulatory elements for efficient tissue or cell specific expression) - broad or selective tropism - viral particle should be small in size to facilitate penetration in tissues - viral prep should be easy to produce in high titers

viral vectors for gene delivery (in vivo)

Adenovirus - double stranded DNA viruses linear genome of app 36 kb, nonenvelopped capsid app 80 nm in diameter

Adeno-associated viruses - non pathogenic, single stranded DNA viruses, virion is a non-enveloped capsid of about 20 nm in diameter, the DNA genome is about 4,7 kb Retrovirus (retro and lentivirus) enveloped RNA viruses with diploid single-stranded genome of 7-11 kb, which reverse transcribed to doube-stranded DNA host cell cytoplasm, the viron size is about 80-100 nm.

Herpes simplex virus-1 enveloped DNA virus, Herpesviridae family, the virion is 120200 nm contains one copy of double-stranded DNA genome of approximately 150 kb

Viral vectors overview

Adeno-associated

Retroviruses And lenti

Adeno

Herpes simplex virus-1

From Bouard Cosset 2009

Comparison of different viral vectors

From Osten Cetin 2007

From wild-type virus to replication defective viral vectors

From Bouard Cosset 2009

Re-targeting option for viral vectors

From Bouard Cosset 2009

Properties of lenti-vectors (consider pseudotyping)

From Bouard Cosset 2009

Lentiviral vector targeting

From Matrai 2010

Lenti

The most studied lentivirus is the human immunodeficiency virus type 1 (HIV-1), which infects CD4- positive T lymphocytes and macrophages (CD4 antigen acts as the primary surface receptor for HIV-1) LTR sequences required for gene expression, reverse transcription and integration

retroviral genome contains three essential genes, termed gag, pol, and env, flanked by long terminal repeats (LTR) Gag gene codes for structural proteins (capsid), matrix, and nucleocapsid, which form the viral core; pol gene codes for viral protease, reverse transcriptase and integrase and env gene codes for envelope glycoproteins Rev is necessary for expression of un-spliced and singly-spliced mRNA in vivo

Basic lentiviral vector

Good capacity Broad tropism Integration long term expression Non toxic, No acute immune response Easy to produce and engineer Self inactivating good for safety TransGene silencing possible Activation of neighboring genes possible (due to activity of LTR)

Principle difference between retro and lenti- vectors is that lenti can transduce quiescent cells From Escors Breckpot 2010

Lentiviral vector production by trans-complimentation

VSV-G has wide tropism but can be toxic for packaging cells From Matrai 2010

Improvement of r-lentivectors

From Escors Breckpot 2010

Lentivector chromosomal targeting

From Matrai 2010

AAV

inverted terminal repeats (ITRs) contain all cis-acting sequences critical for virus packaging, replication, and integration

rep gene contains two promoters and each transcript is regulated by internal splicing, resulting in production of four nonstructural Rep proteins, Rep78, Rep68, Rep52, and Rep40. The Reps regulate replication, viral transcription, packaging of the AAV genomes and sitespecific integration cap gene encodes the three structural proteins, virion protein 1, 2, and 3 (VP1, VP2, and VP3), from two alternative transcripts and an alternative translation initiation codon. The VPs formthe capsid of the virus From Warrington Herzog 2006

AAV

AAV vector trafficking

AAV stays either episomal or integrates into a specific region on the human chromosome 19 (19q13.3-qter), a site termed AAVS1. AAV then stays in a repressed state until reactivated by superinfection with a helper virus

From Warrington Herzog 2006

Modifications of AAV

AAV

Non-pathogenic Simple vector design No inflammatory response Virus is small enabling tissue penetration Long onset of expression (due to the fact that it is a single strand DNA virus requires second strand synthesis, which is often poorly efficient in non proliferating cell Low capacity

adeno

Ad genes are encoded on both DNA strands, and are regulated by complex RNA processing resulting in a high number of viral proteins (for example, over 70 proteins for the well-studied Ad5 serotype)

The early transcription units (E1A,E1B, E2, E3, and E4) function in transcriptional activation and replication of the Ad genome, as well as in subversion of host cell immune response. The late transcription units (L1L5) encode structural proteins for the capsid and internal core. E3-11.6K protein (adenovirus death protein) is produced in the late phase of infection, and aids in the host cell lysis

From McConnell and Imperiale 2006

Generations of adenovectors

helper-dependent (HdAd) Ad vector contains only two flanking ITRs and a packaging sequence ( ). This give high capacity up to 37 kb

replication of HdAd DNA and assembly of virions during virus production is done by coinfectionwith helper adenovirus

From McConnell and Imperiale 2006

adeno
Broad tropism Large capacity No integration but expression has been shown to persist for several months Quick onset of expression (very valuable for in vitro) Relatively difficult to produce Cyto-toxicity Acute immune response

Herpex Simplex Virus

HSV-1 is a common human pathogen; primary infection occurs in childhood via oral mucosa, and leads to lytic mucosal cell death Main advantage very large capacity Ability of HSV vectors to invade and establish lifelong non-toxic latent infections in neurons Three types of vector produced: - vreplication-competent attenuated vectors, - replication-incompetent recombinant vectors and - defective helper-dependent vectors known as amplicons.

Clinical trials
Since 1990, >1500 clinical trials have been approved worldwide utilizing viral and non-viral vectors and targeting various organs, cell types, and diseases A violent innate immune response to an intravenously delivered adenoviral vector caused the death of a patient with a rare metabolic disorder in 1999 Ex vivo retroviral gene transfer of the c-chain (common to several cytokine receptors) to autologous HSC (CD34+ bone marrow cells) cured boys with X-linked SCID. However, 4 of 10 children developed leukemia (Hacein-Bey-Abina et al., 2008) Retroviral transduction of HSC continues to show high therapeutic efficacy in ADA-SCID without the development of leukemia (Aiuti et al., 2009; Aiuti et al., 2002). Eight of ten children have essentially been cured AAV mediated gene transfer of RPE-65 to the retina restored light sensitivity in patients with Leber's congenital amaurosis (LCA) (from Proc Natl Acad Sci USA 105(39):15112-7, 2008

Clinical trials

Basic biology practical approach

Identification of novel genes (proliferation, differentiation, apoptosis etc) Identification of novel combinations of genes Not one gene one phenotype but Many genes one phenotype

Basic biology practical approach

Weber et al., 2009

Thank for your attention

Comparison of different viral vectors (molecular)

Promoters
Cytomegalovirus promoter (CMV) commonly used, good for in vitro but often exhibit mosaic activity in vivo and is not active in some tissues (CNS) Chimeric CMV promoter (CMV + chicken -actine) Elongation factor 1 strong ubiquotous expression Tissue specific promoter (for example for CNS PDGF promoter) Regulatable promoters (Tet ON/OFF)