1. ADENOASSOCIATED VIRUS Basic Characteristics of Adenoassocited Virus: Adeno-associated virusesAdeno-associated viruses, from the parvovirus family, are small nonenveloped viruses (20-25 nm) with a genome of single stranded DNA (ssDNA), which is approximately 4.7 kb in size.genomeDNA These viruses can insert genetic material at a specific site on chromosome 19 with near 100% certainty. chromosome There are a few disadvantages to using AAV, including the small amount of DNA it can carry (low capacity) and the difficulty in producing it. This type of virus is being used, however, because it is non- pathogenic (most people carry this harmless virus).virus In contrast to adenoviruses, most people treated with AAV will not build an immune response to remove the virus and the cells that have been successfully treated with it.adenovirusescells

2. Figure 1. Genome organisation of Adeno-associated viruses. The AAV genome is built of single-stranded deoxyribonucleic acid (ssDNA), either positive- or negative-sensed, which is about 4.7 kilobase long. The genome comprises inverted terminal repeats (ITRs) at both ends of the DNA strand, and two open reading frames (ORFs): rep and cap (see figure 1). The former is composed of four overlapping genes encoding Rep proteins required for the AAV life cycle, and the latter contains overlapping nucleotide sequences of capsid proteins: VP1, VP2 and VP3, which interact together to form a capsid of an icosahedral symmetry. GENOMIC ORGANIZATION OF AAV VECTORS

3. The Inverted Terminal Repeat (ITR) sequences comprise 145 bases each. They were named so because of their symmetry, which was shown to be required for efficient multiplication of the AAV genome. Another property of these sequences is their ability to form a hairpin, which contributes to so-called self- priming that allows primase-independent synthesis of the second DNA strand. The ITRs were also shown to be required for both integration of the AAV DNA into the host cell genome and rescue from it, as well as for efficient encapsidation of the AAV DNA combined with generation of a fully-assembled, deoxyribonuclease-resistant AAV particles. With regard to gene therapy, ITRs seem to be the only sequences required in cis next to the therapeutic gene: structural (cap) and packaging (rep) genes can be delivered in trans. With this assumption many methods were established for efficient production of recombinant AAV (rAAV) vectors containing a reporter or therapeutic gene. LIMITATIONS AND IMPROVEMENTS OF ADENOVIRAL VECTORSGENOMIC ORGANIZATION OF AAV VECTORS

4. Several trials with AAV are on-going or in preparation, mainly trying to treat muscle and eye diseases; the two tissues where the virus seems particularly useful.trials However, clinical trials have also been initiated where AAV vectors are used to deliver genes to the brain. This is possible because AAV viruses can infect non-dividing (quiescent) cells, such as neurons in which their genomes are expressed for a long time.vectors In recent human trials, CD8+ immune cells have recognized the AAV infected cells as compromised and killed these cells accordingly. This action appears to be triggered by part of the capsid or outer coat of the type 2 virus. APPLICATION OF ADENOASSOCIATED VIRAL VECTORS

5. One of the major limitations for the use of AAV as a gene delivery vehicle is the relatively small packaging capacity. The unique ability of AAV vectors to become joined into concatamers by head-to-tail recombination of the ITRs has been exploited as a means to increase the coding capacity. In this approach, either the gene itself or the different elements of the transgene expression cassette are split over two AAV vectors that are administered simultaneously. Transgene expression is obtained only after recombination between the two viral genomes, but the efficiency is often reduced as compared to single vector transduction. The AAV vectors do not contain any viral coding regions, and therefore, there is no toxicity associated with gene expression. However, a single injection of AAV vector elicits a strong humoral immune response against the viral capsid, which will interfere with re- administration of the vector. Furthermore, natural infections have resulted in a high prevalence of circulating neutralizing antibodies against AAV in the majority of the population, which may inhibit transduction. such as the liver-specific albumin promoter, lung-specific cystic fibrosis transmembrane conductance regulator promoter, the cardiac muscle- specific myosin light chain-2 promoter, and the hepatomaspecific - fetoprotein promoter has been described. LIMITATIONS OF ADENOASSOCIATED VIRAL VECTORS

6. Herpes simplex virusesHerpes simplex viruses (HSV) belong to the subfamily of Alphaherpesvirinae. Herpes viruses consists of a relatively large linear DNA genome of double-stranded DNA 150 kb in length, encased within an icosahedral protein cage called the capsid, which is wrapped in a lipid bilayer called the envelope. The envelope is joined to the capsid by means of a tegument. This complete particle is known as the virion.DNAgenomeprotein The genome of Herpes viruses encodes some 100-200 genes. These genes encode a variety of proteins involved in forming the capsid, tegument and envelope of the virus, as well as controlling the replication and infectivity of the virus.virus Herpes simplex virus 1 and 2 (HSV-1 and HSV-2) are two species of the herpes virus family, which cause infections in humans. An infection by a herpes simplex virus is marked by watery blisters in the skin or mucous membranes of the mouth, lips or genitals. The genomes of HSV-1 and HSV-2 are complex, and contain two unique regions called the long unique region (UL) and the short unique region (US). Of the 74 known ORFs, UL contains 56 viral genes,herpes simplex virusgenes HERPES SIMPLEX VIRAL VECTORS

7. HERPES SIMPLEX VIRAL VECTOR whereas US contains only 12. Transcription of HSV genes is catalyzed by RNA polymerase II of the infected host. Immediate early genes, which encode proteins that regulate the expression of early and late viral genes, are the first to be expressed following infection. Early gene expression follows, to allow the synthesis of enzymes involved in DNA replication and the production of certain envelope glycoproteins. Expression of late genes occurs last, this group of genes predominantly encode proteins that form the virion particle. RNAexpression Herpes viruses are currently used as gene transfer vectors due to their specific advantages over other viral vectors. Among the unique features of HSV derived vectors are the very high transgenic capacity of the virus particle allowing to carry long sequences of foreign DNA, the genetic complexity of the virus genome, allowing to generate many different types of attenuated vectors possessing oncolytic activity, and the ability of HSV vectors to invade and establish lifelong non-toxic latent infections in neurons from sensory ganglia from where transgenes can be strongly and long-term expressed.vectors APPLICATION OF HERPES SIMPLEX VIRAL VECTOR

8. AlphavirusesAlphaviruses, like Sindbis Virus and Semliki Forest Virus, belong to the Togaviridae family of viruses. There are 27 alphaviruses, able to infect various vertebrates such as humans, rodents, birds, and larger mammals such as horses as well as invertebrates. Alphaviruses particles are enveloped have a 70 nm diameter, tend to be spherical and have a 40 nm isometric nucleocapsid.viruses The genome of alphaviruses consists of a single stranded positive sense RNA. The total genome length ranges between 11 and 12 kb, and has a 5’ cap, and 3’ poly-A tail. There are two open reading frames (ORF’s) in the genome, non-structural and structural. The first is non structural and encodes proteins for transcription and replication of viral RNA, and the second encodes four structural proteins: Capsid protein C, Envelope glycoprotein E1, Envelope glycoprotein E2, and Envelope glycoprotein E3. The expression of these proteins and replication of the viral genome allgenomeRNAproteins ALPHA VIRUS VIRAL VECTOR

9. takes place in the cytoplasm of the host cells.cytoplasmcells Alphaviruses are of interest to gene therapy researchers, in particular the Ross River virus, Sindbis virus, Semliki Forest virus, and Venezuelan Equine Encephalitis virus have all been used to develop viral vectors for gene delivery. Application of replication-deficient vectors leads to short-term expression, which makes these vectors highly attractive for cancer gene therapy. Alphavirus vectors carrying therapeutic or toxic genes used for intratumoral injections have demonstrated efficient tumor regression.gene therapyvectors Of particular interest are the chimeric viruses that may be formed with alphaviral envelopes and retroviral capsids. Such chimeras are termed pseudotyped viruses. Alphaviral envelope pseudotypes of retroviruses or lentiviruses are able to integrate the genes that they carry into the expansive range of potential host cells that are recognized and infected by the alphaviral envelope proteins E2 and E1. The stable integration of viral genes is mediated by the retroviral interiors of these vectors.pseudotyped viruses retroviruseslentiviruses APPLICATION OF ALPHAVIRUS VIRAL VECTOR

10. There are limitations to the use of alphaviruses in the field of gene therapy due to their lack of targeting, however, through the introduction of variable antibody domains in a non-conserved loop in the structure of E2, specific populations of cells have been targeted. Furthermore, the use of whole alphaviruses for gene therapy is of limited efficacy both because several internal alphaviral proteins are involved in the induction of apoptosis upon infection and also because the alphaviral capsid mediates only the transient introduction of mRNA into host cells. Neither of these limitations extend to alphaviral envelope pseudotypes of retroviruses or lentiviruses. However, the expression of Sindbis virus envelopes may lead to apoptosis, and their introduction into host cells upon infection by Sindbis virus envelope pseudotyped retroviruses may also lead to cell death. The toxicity of Sindbis viral envelopes may be the cause of the very low production titers realized from packaging cells constructed to produce Sindbis pseudotypes. LIMITATION OF ALPHAVIRUS VIRAL VECTOR

11. An alternative to the use of viral vectors for gene delivery is to deliver genetic material in the form of bacterial plasmid DNA. In the simplest form, naked plasmid DNA can be injected into skeletal muscle leading to transfection of muscle fibers close to the site of delivery. Though the transfection efficiency by nonviral vectors is relatively lower than that by viral vectors, synthetic nonviral vectors are designed to overcome many of the problems associated with viral vectors, such as risk of generating the infectious form or inducing tumorigenic mutations, risk of immune reaction, limitation to the size of genes incorporated, and difficulty for the production to scale up. The advantages of nonviral carriers over their viral counterparts are: (1) they are easy to prepare and to scale-up; NON- VIRAL VECTORS

12. (2) they are generally safer in vivo; (3) they do not elicit a specific immune response and can therefore be administered repeatedly; (4) nonviral vectors allow for the delivery of large DNA fragments and are also particularly suitable to deliver oligonucleotides to mammalian cells, which is an excellent feature for the application of antisense strategies to downregulate the expression of certain genes; and (5) they are better for delivering cytokine genes because they are less immunogenic than viral vectors. NONVIRAL VECTORS