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, adenoassociated 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 cellfree 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 of Retroviridae (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

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 Human T-cell leukemiabovine leukemia viral

Moloney murine leukemia virus

simple

4.

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 env region 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-

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 the pol gene. The protease (PR)
is derived from the gag pro gene.

Genomic organization of Retroviruses Simple retroviruses usually carry only the elementary information gag-pol-env, whereas complex retroviruses code for additional regulatory nonvirion proteins derived from multiply spliced messages

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

regions, gag, pro, pol, and env. The pro gene is encoded in the gag reading frame. PBS- tRNAprimer 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 rev RNA binding protein to induce the transition from the early to the late phase of HIV gene expression, tat RNA 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.

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 the pol-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.

(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) (8) (9) (10) Translation: where they are translated by the cellular machinery. Assembly: The full-length RNA and viral proteins are assembled and, Budding: progeny viral particles bud from the plasma membrane, and 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.

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 Internal Ribosome Entry Site (IRES) sequence(I) or introduction of second promoter (P)

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 bacterial gpt gene 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 can be 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 hprt and 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- or tk-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

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 the src 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 the src 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; DRdirect repeat;

LTR- long terminal repeat

Advantages of Retroviral Vectors Retroviral vectors have the ability to transduce a wide range of cell types from different animal 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 by proteins 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 for in vivo transduction into pulmonary or renal cells which have quite silent cell turnover Integration has been associated with oncogene activation after transduction of hematopoietic cells It is difficult to prepare high titres of viral stocks, and the retrovirus cannot carry very large 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 from different 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 expression
are 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),

Cell lineage analysis- Retroviral vectors provide useful tools for cell lineage analysis because 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 wide
range of cell types, including primary cells, providing an efficient means to transfer and express the cDNAs.

Immunoglobulin rearrangement- by constructing retroviral vectors that contain sequences

from 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 mark
individual 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 somatic
cells, 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 the
expression 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 infect dividing cells without producing any immunogenic viral proteins while also becoming a permanent part of the host cell.

Studies of bone repair- used to deliver various growth factors and differentiation factors to 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) in infants and preadolescents. Lentiviral vectors can infect mouse and rat embryos to generate transgenic animals with high 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 further classified 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 shell70100 nm in diameter. Capsid-12 identical copies of the trimeric hexon 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 inverted terminal repeats (ITRs) of 100-140bps

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 E1A E1B Function Activates early-phase transcription and induces the S phase of the host cell 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 E4 Codes for proteins that block natural cellular responses to viral infection 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,

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 genome enters 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

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 Adenoviral 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 need not 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 in adenoviral 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 appropriate surface 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

Preclinical Gene Transfer and Clinical Trials Studies of cancer treatment- successfully delivered tumor suppressor genes p53 and p16 to tumor growths. Suicide gene therapy, or prodrug therapy, has also been studied as a cancer treatment option. 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 diseases because of the vectors ability to affect nondividing cells and its high concentration in the liver after administration. A recent study has assessed the 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 pulmonary tuberculosis. Biosafety measures using viral vectors Requires implementation of biosafety level-2 containment (BSL-2) practices. Protocol must 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 gene infects rodent cells, amphotropic env gene infects murine and non-murine cells, including human. Mode of Transmission: by direct contact or blood-borne.

Risks associated with Adenoviral vectors Can be infective and quite stable - after extracting with ether and/or chloroform unlike HIV 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. Recombinant Adenovirus vectors infect a variety of mammalian cell types. Mode of Transmission: by droplet, aerosol, injection

Laboratory 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 heat inactivation. Severely immunocompromised individuals should not go into a room with retroviral agent. 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
Insert capacity Virus Retroviruses Adenovirus Genome ssRNA 712 kb dsDNA 36 kbp (kb) 7 8 Specific Long-term RNA Titer 106107 10111012

integration maintenance intermediate Y N Y N Y N

Insert capacity Virus Genome (kb) 4.5 25

Specific

Long-term

RNA Titer 106108 1091010

integration maintenance intermediate Y N Y Y N N

Adeno-associated virus ssDNA 4.7 kb Herpes simplex virus dsDNA 150 kbp

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 Harbor Laboratory Press (NY). William C. Heiser (Editor), Methods in Molecular Biology-Gene Delivery to mammalian cells, Vol 2, Viral gene transfer techniques, Vol 246, Humana Press. Otto-Wilhelm Merten and Mohamed Al-Rubeai (eds.),(2011),Viral Vectors for Gene Therapy: Methods and Protocols, Methods in Molecular Biology, vol. 737 ,Springer, Science+Business Media, LLC.