Genome Structure of Retroviruses LTR LTR U3 R U5 U3 R U5 Essential characteristics of a simple retrovirus: 1. The viral DNA contains large redundant sequences at the two ends of the genome designated long terminal repeats (LTRs). LTRs can be further divided into U3 (unique 3), R (repeat), and U5 (unique 5) regions. U3: The viral promoters and transcriptional enhancers are located in the U3 region; R: The R region is essential for reverse transcription and replication of all retroviruses. In addition, the R regions of some viruses also contain elements important for gene expression. U5: The U5 region contains sequences that facilitate the initiation of reverse transcription. 2. Immediately downstream of the 5’ LTR is a primer binding site (PBS) that has sequence complementarity to a portion of a cellular tRNA. Different tRNAs are used by different viruses as primers for the initiation of reverse transcription.

Essential characteristics of a simple retrovirus: 3. The packaging signal (C) or encapsidation signal (E) are sequences that interact with the viral proteins to accomplish specific packaging of the viral RNA. 4. The coding regions of all retroviruses contain at least three genes. Gag: The gag gene near the 5’ end of the viral genome codes for Gag polyproteins that make up the viral capsid. The pol gene encodes reverse transcriptase and integrase. Reverse transcriptase copies the viral RNA to generate the viral DNA, whereas integrase integrates the viral DNA into the host chromosome to form a provirus. The env gene codes for the envelope polyprotein, which is cleaved into the transmembrane domain and the surface domain (SU). The sequences that encode the viral protease (Pro) are always located between gag and pol and are most often expressed as either a part of the Gag polyprotein or as a part of the Gag-Pol polyprotein. 5. The region between env and the 3’ LTR contains a purine-rich region known as polypurine tract (PPT) that is important for reverse transcription. 6. Short sequences at the two ends of the LTR are important for integration and are referred to as attachment sites (att). The att interact with integrase and are necessary for efficient integration of the viral DNA.

DEVELOPMENT OF RETROVIRAL VECTOR The first retroviral vector systems were derived from murine leukaemia virus (MLV). There were (and are still) a number of reasons for choosing MLV as the basis for such gene delivery systems, including (i) the biology of this retrovirus is particularly well understood, (ii) the MLV genome was among the earliest retroviral genomes molecularly cloned and (iii) these viruses are able to infect cells efficiently. Retroviral vector systems consist of two components: (i) a vector construct that carries the gene to be delivered and provides the genome for the recombinant virus, and (ii) a cell line that provides the viral proteins required to produce the recombinant virus, known as packaging cells Construction of vector -Retroviral vectors are constructed from the proviral form of the virus. The gaga, pol, and env genes are removed by the selectable marker to make room for the gene(s) of therapeutic interest and to eliminate the replicative functions of the virus. Upto 8 Kilobases of heterologous DNA can be incorporated into the retroviral vector.

Construction of vector -Along with the gene of therapeutic interest, gene encoding antibiotic resistance often are included in the recombibant as a means of selecting the virus-harbouring cultured cells. For example, aminoglysoside-3’-phosphotransferase gene for kanamycin, hygromycin B transferase gene for hygromycin. The antibiotic resistance gene confers resistance to antibiotics. - Sequences containing promoter and enhancer functions may also be included with the transgene to facilitate the efficient expression, and in some circumstances, to provide for tissue-specific expression after administration in vivo. Alternatively, the promoter and enhancer functions contained in the LTR of the virus may be used for this purpose. Marker gene Therapeutic gene with separate internal promoter to drive high level expression LTR NeoR CMV ADA LTR

Packaging Cell Lines or Helper Cell To produce recombinant retroviral vector virions, the vector construct carrying the gene(s) to be delivered is introduced by physical gene transfer methods (such as transfection, electroporation etc.) into a retroviral packaging cell line or helper cell. Helper cells are engineered culture cells expressing viral proteins needed to propagate retroviral vectors; this is generally achieved by transfecting plasmids expressing viral proteins into culture cells. Most helper cell lines are derived from cell clones to ensure uniformity in supporting retroviral vector replication. These helper or packaging cells produce the viral structural (Gag and Env) proteins and enzymes (pol-encoded RT, IN), but are not able to package the viralRNA encoding these proteins since the region required for encapsidation has been deleted. Instead the proteins recognise and associate with genomic length RNA from the introduced vector construct, which carries an intact region, to form recombinant virus particles. The recombinant virus particles carrying the retroviral vector genome bud out of the packaging cell line into the cell culture medium. The virus-containing medium is either directly filtered to remove cells and cellular debris and then used to infect the target cell, or virus is purified and concentrated before infecting target cells.

Major Problem with Retroviral Vector After the virus has bound to the receptor on the cell surface, the viral capsid is delivered into the cell and the viral RNA is reverse transcribed into a DNA form which integrates into the host cell DNA. The integrated viral DNA (provirus) functions essentially as any other cellular gene and directs the synthesis of the products of the delivered gene(s). Major Problem with Retroviral Vector The major problem with two-component retroviral vector systems arises as a result of the naturally occurring phenomenon of homologous recombination. If the vector provirus and the provirus providing the structural proteins in the packaging cells recombine, there is a possibility that replication- competent retrovirus will arise. Such virus is essentially a wild-type retrovirus and no longer carries the delivered gene(s). Replication-competent virus rapidly infects many cells and may eventually cause insertional mutagenesis. Consequently, considerable effort has been devoted to the design of superior packaging systems that drastically reduce the possibility of recombination occurring, as well as to produce improved, safer vectors that cannot replicate even if recombination occurs.

IMPROVEMENTS TO PACKAGING CELLS 1. Improvements to packaging cells have involved removing as much of the retroviral information as possible to reduce the possibility of homologous recombination occurring. The retroviral promoter and termination sequences can be replaced by heterologous promoters and termination sequences. This has the additional advantage of allowing the use of promoters that are more strongly active than the retroviral promoter, thereby giving rise to higher levels of viral protein production. 2. The coding information for the viral proteins cannot be removed by necessity, but these proteins can be made from separate constructs so that additional recombination events are required to recreate a complete replication-competent retrovirus. This has been achieved by expressing the Gag and Pol proteins from one construct and the Env proteins from a second construct. 3. In addition to the improvements to packaging cells, safer retroviral vector constructs also have been produced that carry an artificially inserted stop codon in the Gag reading frame. This ensures that even if replication-competent virus is generated, it will not be able to express its Gag and Pol proteins and thus virus assembly and release will be inhibited.

CLINICAL ADMINISTRATION OF RETROVIRUS The clinical administration of retroviruses has been accomplished by: - Ex vivo transduction of patients cell, - Direct injection of virus into tissue, - Administration of retroviral producer cells. 1. Ex vivo gene transfer: The ex vivo approach has been most widely employed in human clinical trials. Although cumbersome in that it requires the isolation and maintenance in tissue culture of the patients cell. It has the advantage that the extent of gene transfer can be quantified readily and a specific population of cells can be targetted. 2. In vivo gene transfer: Retroviruses are being tested as potential agents to treat brain tumors which, in many circumstances, are relatively inaccessible. Although the direct stereotactic injection of recombinant retrovirus into the target tissue is possible, the efficiency of gene transfer is very low.