retro and adeno virus mediated gene transfer

7/30/2019 Retro and Adeno Virus Mediated Gene Transfer

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Retro and Adeno Virus mediated gene transfer

Viruses have complex and precise structural features, which have adjusted through

natural evolution for efficient transfection of specific host cells or tissues. Viral vector is the

most effective means of gene transfer to modify specific cell type or tissue and can be

manipulated to express therapeutic genes. Several virus types are currently being investigated for

use to deliver genes to cells to provide either transient or permanent transgene expression. These

include adenoviruses (Ads), retroviruses (g-retroviruses and lentiviruses), poxviruses, adeno-

associated viruses, baculoviruses, and herpes simplex viruses.

Retro virus mediated gene transfer

Discovery of Retroviruses

Retroviruses were discovered in 1908, the Danish physician-veterinarian team of Vilhelm

Ellermann and Oluf Bang showed that chicken leukosis, a form of leukemia and of lymphoma,

was caused by a virus. These agents are now known under the collective term avian leukosis

virus or ALV. In 1911, Peyton Rous at the Rockefeller Institute in New York reported the cell-

free transmission of a sarcoma in chickens known as Rous sarcoma virus. Together, these viruses

constitute the avian C-type virus genus, often referred to as avian sarcoma/ leukosis viruses

(ASLV).

Classification of Retroviruses

Retroviruses comprise a large and diverse family of enveloped RNA viruses defined by common

taxonomic denominators that include structure, composition, and replicative properties. The

family ofRetroviridae (reverse transcriptase oncoviruses) is classified into the two

subfamilies; Orthoretroviriniae and Spumaretrovirinae. The first includes six genera (-, -, -,

- and e-retroviruses, and lentiviruses); the genus foamy virus belongs to the latter subfamily.

The first five genera represent mostly simple retroviruses. Because they possess oncogenic

potential, they were formerly referred to as oncoretroviruses. Lenti- and spumaviruses are

complex retroviruses. Distinguishable by the organization of their genomes, retroviruses are

broadly divided into two categoriessimple and complex


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Table: Classification of Retroviruses

Genus Example Genome

1. Avian sarcoma and leukosis viral group Rous sarcoma virus simple

2. Mammalian B-type viral group mouse mammary tumor virus simple

3. Murine leukemia-related viral group Moloney murine leukemia virus simple

4. Human T-cell leukemiabovine leukemia viral human T-cell leukemia virus complex

5. D-type viral group Mason-Pfizer monkey virus simple

6. Lentiviruses human immunodeficiency virus complex

7. Spumaviruses human foamy virus complex

Morphology of Retroviruses

The virions are 80100 nm in diameter, and

their outer lipid envelope incorporates and

displays the viral glycoproteins. The viral

envelope is formed by a cell-derived lipid bilayer

into which proteins encoded by the envregion of

the viral genome are inserted. These consist of the

transmembrane (TM) and the surface (SU)

components linked together by disulfide bonds.

Internal nonglycosylated structural proteins form

the capsid core protein having a icosahedral

structure encoded by the gag region of the viral

genome. It enclosed the virion RNA which is 7

12 kb in size, and it is linear, single-stranded,

nonsegmented, and of positive polarity. The hallmark of the family is its replicative strategy

which includes as essential steps reverse transcription of the virion RNA into linear double-


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stranded DNA and the subsequent integration of this DNA into the genome of the cell. Also

contained in the core are three enzymes, protease (PR), reverse transcriptase (RT), and integrase

(IN), which are derived by proteolysis of a polyprotein product of thepolgene. The protease (PR)

is derived from the gag pro gene.

Genomic organization of Retroviruses

Simple retroviruses usually carry only the elementary informationgag-pol-env, whereas complex

retroviruses code for additional regulatory nonvirion proteins derived from multiply spliced

messages

(A) A simple retrovir al genome-The genetic map of an MMLV contains four major coding

regions,gag,pro,pol, and env. Thepro gene is encoded in thegagreading frame. PBS- tRNA-

primer binding site. The terminal noncoding sequences (long terminal repeats-LTR) include two

direct repeats (R), a U5 (5'unique), and a U3 (3'unique) sequence, - psi-defines packaging

sequence

(B) A complex retroviral genome- The genetic map of HIV contains, besides the major coding

domains, information for other regulatory proteins like revRNA binding protein to induce the

transition from the early to the late phase of HIV gene expression, tatRNA binding protein that

enhances transcription, nef- Disturbs T-cell activation and stimulates HIV infectivity, vpr -

Mediates HIV to infect nondividing cells, vpu- Enhances the release of HIV-1 from the cell

surface to the cytoplasm, vif- A polypeptide necessary for the replication of HIV-1.


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Retrovirus life cycle

(1) Binding and (2) Fusion: Interaction of the glycoprotein with a cellular receptor molecule

is necessary for the successful infection of a cell.This entry into the cell represents the first step

of the viral infection cycle interaction of the viral envelope proteins with the host cell surface

receptors and loss of the envelope by fusion of the virus membrane with the cell membrane.

(3) Uncoating and reverse transcription: After fusion of the lipid membranes of the virus

and host cell, the genetic material of the virus and thepol-encoded proteins are transferred to the

cytoplasm. Viral RNA is converted into DNA by reverse transcriptase. The sequences at the ends

of the viral RNA, the long terminal repeats (LTRs), are duplicated and added at both ends of the

newly synthesized DNA.

(4) Nuclear entry: The dsDNA enters into the host cell nucleus. However, nuclear entry

requires cell division for MLV and ALV. It can only integrate into the host genome of a cell that

are undergoing mitosis, but is an active process for HIV.


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(5) Integration: After nuclear translocation, the viral DNA is integrated into the host

genomic DNA by the integrase, and is then termed 'provirus'.

(6) Transcription: Proviral DNA transcription is performed and mRNA molecules

transferred into the cytoplasm,

(7) Translation: where they are translated by the cellular machinery.

(8) Assembly:The full-length RNA and viral proteins are assembled and,

(9) Budding:progeny viral particles bud from the plasma membrane, and

(10) Maturation: further mature into infectious a particle which repeats the life cycle.

The genomic structure of(A) an integrated provirus and its (B) transcription and (C) translation

products.

A. The viral genome consisting of the gag, pol, and env genes with the long terminal repeats

(LTRs) subdivided into the U3, R, and U5 regions. SD and SA are splice donor and acceptor

sites which defines the packaging sequence.

B. Transcription- This integrated DNA is transcribed into both full length and spliced transcript.C. Translation- A Gag and Gag-Pol polypeptide are translated from the full genomic transcript,

while the envelope (Env) protein is translated from the subgenomic transcript. Gag polyprotein

has myristate modification on its N-terminus, encodes capsid core.


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Retroviral Vector Derivation

Retroviral vectors are typically used to induce the production of a specific protein in transduced

cells. Most replication-defective retroviral vectors have been derived from the murine and avian

retroviruses. Vector design involves the construction of a provirus that contains all of the

signals needed in cis for vector packaging, reverse transcription, and integration, but which lacks

the coding regions for most or all of the viral proteins, and a corresponding packaging cell line to

produce the viral proteins required for viral assembly and transduction. Although it is in

principle possible to derive vectors from any retrovirus, the complexity, toxicity, and regulatory

intricacies of some retroviruses, especially the lentiviruses such as HIV, make construction of

vectors more difficult, and the titers of such vectors are typically low. Since lentivirus can infect

nondividing cells, the additional work needed to develop efficient vectors based on these viruses

appears to be justified. The simplest approach is to use the promoter in the retroviral LTR to

control the expression of a cDNA encoding the protein of interest. Changes can be made in the

enhancer/promoter of the LTR to provide tissue-specific expression or inducibility. The presence

of retroviral sequences between the viral promoter and the translational start codon of the

inserted gene appears not to markedly affect gene expression, even though several ATG start

codons are present in the 5-untranslated regions of commonly used vectors. Alternatively, a

single coding region can be expressed by using an internal promoter.

A. Schematic representation of MMLV-gag-pol-env flanked by LTRs B. Vector construct with deletion of major viral genes retaining LTRs and psi ()

sequence

C. More than one gene product or sequence can be expressed by use of an InternalRibosome Entry Site (IRES) sequence(I) or introduction of second promoter (P)


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Selectable Markers

Many different selectable markers have been used successfully in retroviral vectors. These

include the bacterial neomycin and hygromycin phosphotransferase genes which confer

resistance to G418 and hygromycin, respectively; a mutant mouse dihydrofolate reductase gene

(dhfr*) which confers resistance to methotrexate; the bacterialgptgene which allows cells to

grow in medium containing mycophenolic acid, xanthine, and aminopterin; the

bacterial hisD gene which allows cells to grow in medium without histidine but containing

histidinol; the multidrug resistance gene (mdr) which confers resistance to a variety of drugs; and

the bacterial genes which confer resistance to puromycin or phleomycin. All of these markers are

dominant selectable markers and allow chemical selection of most cells expressing these genes.

-galactosidase can also be considered a dominant marker; cells expressing -galactosidase canbe selected by using the fluorescence-activated cell sorter. In fact, any cell surface protein can

provide a selectable marker for cells not already making the protein. Cells expressing the protein

can be selected by using a fluorescent antibody to the protein and a cell sorter.

Other selectable markers that have been included in vectors include the hprtand HSV

thymidine kinase, which allow cells to grow in medium containing hypoxanthine, amethopterin,

and thymidine. For these selectable markers to be useful, however, the target cells must initially

be hprt- ortk-deficient.

Types of replication vectors

Retroviral vectors can either be

replication-defective or replication-competent

Replication-defective vectors:

Replication-defective vectors are the most common choice in studies. They are capable of

infecting their target cells and delivering their viral payload, but then fail to continue the typical

lytic pathway that leads to cell lysis and death. This is accomplished by replacing most or all of


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the coding regions of a retrovirus with the gene(s) or sequence elements to be transferred, so that

the vector by itself is incapable of making proteins required for additional rounds of viral

replication and packaging. Viral proteins needed for the initial infection can be provided

in trans by a retroviral packaging cell; there is no need for retroviral protein synthesis in

recipient cells for proviral integration. The process of gene transfer and expression by retroviral

vectors is often referred to as transduction rather than infection to differentiate this process from

productive viral infection by a replication-competent virus. Depending on the viral vector, the

typical maximum length of an allowable DNA insert in a replication-defective viral vector is

usually about 8-10 kB. While this limits the introduction of many genomic sequences, most

cDNA sequences can still be accommodated.

Replication-competent viral vectors:

Conversely, replication-competent viral vectors contain all necessary genes for virion

synthesis, and continue to propagate themselves once infection occurs. Because the viral genome

for these vectors is much lengthier, the length of the actual inserted gene of interest is limited

compared to the possible length of the insert for replication-defective vectors.

For example, in Avian viruses, Rous sarcoma virus (RSV) carries thesrc oncogene in

addition to a full complement of genes required for replication, and thus provides the earliest

example of a replication-competent retroviral vector. RSV has been exploited as a vector by

replacement of thesrc gene with other cDNAs. Vectors derived from RSV efficiently infect

avian cells, and although they can infect mammalian cells, the efficiency of infection is quite

low. Recently, the receptor for subgroup A RSV has been cloned whose expression in

mammalian cells allows the efficient infection of those cells by the RSV(A)-based vectors.

SD- Splice donor;

SA- splice acceptor;

DR- direct repeat;

LTR- long terminal

repeat
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Advantages of Retroviral Vectors

Retroviral vectors have the ability to transduce a wide range of cell types from differentanimal species,

Used to integrate genetic material carried by the vector into recipient cells precisely, Used to express the transduced genes at high levels, They lack the spread of vector or production of viral proteins after infection, In contrast to typical vectors derived from oncoviruses, lentiviruses such as human

immunodeficiency virus (HIV) useful for gene therapy applications, especially those

involving in vivo gene transfer or transfer to slowly dividing cells such as hematopoietic

stem cells.

Disadvantages of Retroviral Vectors

They are generally not useful for systemic administration because of their inactivation byproteins and cellular components of human blood, thus used to transduce cells ex vivo

They require cell division for efficient integration and thus not a very feasible vector forin vivo transductioninto pulmonary or renal cells which have quite silent cell turnover

Integration has been associated with oncogene activation after transduction ofhematopoietic cells

It is difficult to prepare high titres of viral stocks, and the retrovirus cannot carry verylarge amounts of transgenic material

Expression of the transgene is difficult to control

Experimental Applications

Constitutive gene expression- Can efficiently transduce a wide variety of cells types fromdifferent species and to induce high levels of protein expression.For example, large

cDNAs such as the insulin like growth factor I receptor (4.1-kb coding region) have been

transmitted efficiently and expressed at high levels (>106

molecules per cell) in

transduced cells.

Regulated gene expression-Retroviral vectors designed to provide regulated gene expressionare quite useful in the context of gene therapy, where it may be important to limit expression to

specific cell types (e.g., red cells for globin), to regulate the overall level of expression (e.g., for

clotting factors),


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Cell lineage analysis- Retroviral vectors provide useful tools for cell lineage analysisbecause they integrate into the genome of the transduced cell and remain stable during

cell division and differentiation. Retroviral vectors have been used to transduce neuronal

cells in vivo in studies designed to follow the fates of the marked cells.

cDNA library construction-The vectors can be used to transfer and express cDNAs in a widerange of cell types, including primary cells, providing an efficient means to transfer and express

the cDNAs.

Immunoglobulin rearrangement- by constructing retroviral vectors that contain sequencesfrom the immunoglobulin locus and studying the rearrangement of these sequences after the

vector has been introduced into appropriate cells

Transgenic animals- important in species where direct DNA transfer is difficult. For example,the reproductive physiology of chickens has made the direct injection of DNA into fertilized eggs

quite difficult.

Chromosome tagging-Retroviral vectors carrying selectable genes can also be used to markindividual chromosomes.

Shuttle vectors- Origins of replication and drug resistance genes from bacterial plasmids(amp and neo) have been included in the vector to facilitate the cloning of vector and

flanking sequences from cells containing integrated vectors.

Cellular immortalization- the methods for generating immortal cell lines from the somaticcells, for example, infection of human fibroblasts with SV40, are relatively inefficient. Retroviral

vectors expressing the SV40 T antigen are more efficient.

Antisense RNA and Ribozyme production- Retroviral vectors have been used to inhibit theexpression of specific genes by the inclusion of transcriptional units intended to induce the

synthesis of antisense RNA or specific ribozymes.

Therapeutic Applications

Preclinical Gene Transfer and Clinical Trials

Studies of tissue repair and engineering- Because these vectors can be used to infectdividing cells without producing any immunogenic viral proteins while also becoming a

permanent part of the host cell.


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Studies of bone repair- used to deliver various growth factors and differentiation factorsto both mature bone cells and stem cells that have been used in tissue scaffolding, and

used in repair of damaged cartilage and in the formation of tissue-engineered blood

vessels for the treatment of cardiovascular disease.

Used in clinical trials, to treat X-linked severe combined immunodeficiency (X-SCID) ininfants and preadolescents.

Lentiviral vectors can infect mouse and rat embryos to generate transgenic animals withhigh tissue specific expression of transgene.

Adenoviruses mediated gene transfer

First discovered by Wallace Rowe and his colleagues in human adipose tissue (1953) Family Adenovirideae- which is divided into two genera: the Aviadenovirus genus and

Mastadenovirus

Human Adenoviruses are furtherclassified into six species (AF). subdivided into over 50 infective serotypes- recombinant adenoviral vectors are based on

human adenovirus serotypes 2 (Ad2) and 5 (Ad5) of subgroup C and are most effective.

These serotypes cause a mild respiratory disease in humans and are non-oncogenic,

Morphology of Adenoviruses Non-enveloped, icosahedral protein shell-

70100 nm in diameter.

Capsid-12 identical copies of the trimerichexon protein, pentameric penton base

protein is located at each vertex of the

capsid, from it extends a trimeric fiber

protein that terminates in a globular knob

domain.

Genome- linear, dsDNA (26-40kb), compact nucleosome like structure having invertedterminal repeats (ITRs) of 100-140bps


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Genomic organization Adenoviruses

Viral genome comprises two major transcription regions, the early region (E1, E2, E3,

and E4- transcription units) and the late region (L1-L5).

Transcription

unit Function

E1A Activates early-phase transcription and induces the S phase of the host cell

E1B Codes for E1B 19K and E1B 55K, which inhibits apoptosis and allow for viral

replication

E2 Codes for DNA polymerase (pol), preterminal protein (pTP), and DNA binding

protein (DBP)

E3 Codes for proteins that block natural cellular responses to viral infection

E4 Codes for a variety of proteins that perform in DNA replication, mRNA transport,

and splicing

Adenoviral life cycle

The early phase of adenoviral DNA invasion begins when the virus comes in contact with a host

cell and ends at the onset of DNA replication. The globular knob domain of the viral capsid has a

high affinity for the coxsackie virus and adenovirus receptor (CAR), which can be found on a

variety of cells throughout the human body. When the virus locates a host cell, the process of

binding and internalization begins. The virushost cell affinity between the fibrous knob and the

CAR is heightened by the interaction of the penton base protein with secondary cellular

receptors. The virus then travels through the cell membrane via receptor-mediated endocytosis,


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the virion is released, and the genome escapes the protein capsid and makes its way into the host

cell nucleus, as depicted in Figure.

The virion then escapes from the endosome and localizes to the nuclear pore, whereupon its

genome is translocated to the nucleus. Transcription of viral DNA begins when the genomeenters the host cell nucleus. At this time, the E1A transcription unit of the early phase is

transcribed, followed quickly by the E1B unit. Together, these two units help to prepare the

genome for further transcription, shift the host cell into the S phase of replication, and inhibit

apoptosis of the host cell. The E2 unit, the next to transcribe, encodes for DNA polymerase, a

preterminal protein, and a DNA-binding protein, all of which are necessary for DNA replication.

This process is followed by the transcription of the E3 unit, which inhibits the host cell from

responding to the viral invasion. Finally, the E4 unit is transcribed to produce a variety of

proteins required for DNA replication and movement into the late phase. The late phase begins at

the onset of viral DNA replication. This process begins at the origins of replication in the ITRs

on either end of the viral genome, and the terminal protein at each end of the chromosome acts as

the DNA primer. The products of late-phase transcription are expressed after a 20-kb section of

the major late promoter has been transcribed. This section then undergoes multiple splicing


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cycles to return five encoding proteins of the late mRNA. These proteins are later used either to

form the viral capsid or to assist in assembling the viral progeny. The host cell finally

disintegrates and the virus is released.

Design and Construction of Adenovir al Vectors

The first-generation vectors are the most commonly used viral vectors in gene-therapy trials.

These vectors, based on Ads 2 and 5 of species C, have the E1 region of the genome deleted to

allow more genomic space for foreign DNA. The E3 region may also be deleted for viral DNA to

be replicated in culture. These eliminations allow the insertion of approximately 7.5 kb of DNA

into the vector. Another vector form used in gene therapy is known as the gutted vector, in

which all adenoviral DNA is excised except for the ITRs and packing signals. These vectors

allow up to 36 kb of foreign DNA to be accommodated within the viral vector.

Advantages of adenoviral vectors

Adenovirus does not go through an RNA intermediate, and thus inserted sequences neednot be compatible with transcription of the complete viral genome and its subsequent

reverse transcription as for retroviral vectors.

With a genome size of 36 kb, there is considerable room for additional DNA inadenoviral vectors.

They have the ability to infect both dividing and quiescent cells Adenoviral recombinant vectors are stable and non-oncogenic Vector titers can be very high, up to 1012 transducing units per milliliter.

Disadvantages of adenoviral vectors

Possess tropism of the parent viruses which can infects all cells that possess appropriatesurface receptors, which precludes the targeting of specific cell types

Long-term correction not allowed Humoral and cellular immune response from high vector doses

Therapeutic Applications


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Preclinical Gene Transfer and Clinical Trials

Studies of cancer treatment- successfully delivered tumor suppressor genes p53 and p16to tumor growths.

Suicide gene therapy, or prodrug therapy, has also been studied as a cancer treatmentoption. Suicide therapy uses viral proteins to metabolize nontoxic drugs into a toxic form,

resulting in cell death. Recently, a phase I/II suicide-gene-therapy clinical trial has been

completed in prostate-cancer patients, using an E1/E3-deleted replication deficient Ad

(CTL102) encoding the bacterial nitroreductase enzyme in combination with prodrug

CB1954.

Study of various liver diseasesbecause of the vectors ability to affect nondividing cellsand its high concentration in the liver after administration. A recent study has assessedthe therapeutic effect of an Ad vector carrying PAI-1 small interfering RNA (siRNA) on

hepatic fibrosis.

Studies of stem cell differentiation, AIDS, cardiovascular disease, and pulmonarytuberculosis.

Biosafety measures using viral vectors

Requires implementation of biosafety level-2 containment (BSL-2) practices. Protocolmust be approved by the Institutional Biosafety Committee (IBC)

Risks associated with Retroviral vectors

Generation of replication competent retroviruses (RCR) in target cells or tissues Increase in target cell range of the vector increases the risk of RCR Nature of the vector coding sequence-genes involved in oncogenesis, growth regulation,

innate or adaptive immunity, or infectious diseases carry a greater risk

Host Range: dependent on the specificity of the viral envelope- ecotropic env geneinfects rodent cells, amphotropic env gene infects murine and non-murine cells, including

human.

Mode of Transmission: by direct contact or blood-borne.


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Risks associated with Adenoviral vectors

Can be infective and quite stable - after extracting with ether and/or chloroform unlikeHIV or herpes

does not integrate into the host cell genome but can produce a strong immune response Possess tropism of the parent viruses- infects all cells that possess appropriate surface

receptors, which precludes the targeting of specific cell types

Host Range: Humans are the natural reservoir for wild type Adenovirus 5. RecombinantAdenovirus vectors infect a variety of mammalian cell types.

Mode of Transmission:by droplet, aerosol, injectionLaboratory Practices

Usage of eye protection, masks, gloves, Biosafety cabinets Vacuum lines must be fitted with a hydrophobic and HEPA filter. Biohazard waste containers should be disinfected and autoclaved before disposing. Most effective germicides Phenol (5%), Sodium hypochlorite (household bleach diluted

to 200 ppm or 10%) with a minimum 15 mins contact time must be used.

Viral vectors must be tested for presence of replication competent viruses after heatinactivation.

Severely immunocompromised individuals should not go into a room with retroviralagent.

No standard tests and/or surveillance methods are available to determine possible

viral vector infection although serious illness can be managed by treating symptoms and

complications of the infection.

Characteristics of Viruses Used to Derive Gene Transfer Vectors

Virus Genome

Insert capacity

(kb)

Specific

integration

Long-term

maintenance

RNA

intermediate Titer

Retroviruses ssRNA 712 kb 7 Y Y Y 106107

Adenovirus dsDNA 36 kbp 8 N N N 10111012


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Virus Genome

Insert capacity

(kb)

Specific

integration

Long-term

maintenance

RNA

intermediate Titer

Adeno-associated virus ssDNA 4.7 kb 4.5 Y Y N 10 10

Herpes simplex virus dsDNA 150 kbp 25 N Y N 10 10 The ability of viruses to deliver foreign DNA to cells for therapeutic purposes has been

exploited in numerous different contexts. The diverse nature of different vectors and the

variability of different diseases mean that there will almost certainly be no one size fits all

vector. Clinical trials have shown that certain vectors have great potential for specific diseases.

For example, retroviral vectors have had great success in treating X-SCID, whereas lentiviral

vectors have been used to target various neurological diseases, including Parkinsons and ALD,

and other clinical trials have employed AAV vectors to treat monogenic disorders, such as

Duchenne muscular dystrophy and hemophilia B. Although no viral vector has yet received

clinical approval in Europe or the USA, the encouraging results from clinical trials, coupled with

continual improvements in vector design and safety, shows that this technology has immense

potential.

References:

Coffin JM, Hughes SH, Varmus HE, editors. (1997),Retroviruses. Cold Spring HarborLaboratory Press (NY).

William C. Heiser (Editor), Methods in Molecular Biology-Gene Delivery to mammaliancells, Vol 2, Viral gene transfer techniques, Vol 246, Humana Press.

Otto-Wilhelm Merten and Mohamed Al-Rubeai (eds.),(2011),Viral Vectors for GeneTherapy: Methods and Protocols, Methods in Molecular Biology, vol. 737 ,Springer,

Science+Business Media, LLC.

retro and adeno virus mediated gene transfer

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