Adenovirus-based Gene Therapy: a Promising Novel Cancer Therapy

A Viral Biorealm page on the family Adenovirus-based Gene Therapy: a Promising Novel Cancer Therapy

[Image:Genetherapy.jpeg|thumb|500px|left| The retrovirus infection cycle. [1].]]

Baltimore Classification

Group I, double stranded DNA

Higher order categories

Order Picrnovirales
Family Adenoviriade
Genera Atadenovirus, Aviadenovirus, Ichtadenovirus, Mastadenovirus, Siadenovirus

Adenovirus (Ad) structure is characterized by its medium size (90-100nm) and non-enveloped icosahedral, including a nucleocapsid. It is the largest of the non-enveloped viruses, and contains a ds DNA genome. About 57 serotypes exist in humans and cause 5-10% of upper respiratory infections in children; it is also highly prevalent among adults. Ad infects various species of vertebrates and were first found and isolated in human adenoids in the mid 1950s.

Ad is an extremely tough virus, ubiquitous in human and animal populations as it can survive long periods of time out of the host and is endemic year round. Infection has been found in multiple organ systems, yet most remain asymptomatic. Most adults have measurable titers of anti-Ad antibodies, indicating a prior infection or exposure. The virus is oncogenic in rodents only.

Respiratory tract infections are a result from droplet inhalation, while gastrointestinal problems are due to fecal-oral transmission. There are 3 different types of interactions that can occur upon infection. Lytic infection occurs when the adenovirus enters human epithelial cells and continues through an entire replication cycle, with a host inflammatory response. Chronic or latent infection can also occur, infecting the lymphoid tissue, and involves no symptoms. The exact mechanism is unknown. The last interaction that can occur is oncogenic within rodents, as an adenovirus integrates its own DNA into host DNA and produces E1A proteins, altering transcription, deregulating apoptosis and forming malignant tumors. E1A facilitates oncogeny by activation of ras or c-src signaling, polyoma middle T protein. Paradoxically, this role of E1A is not seen in humans, as Ad sequences are non-existent in human tumor cells, despite being highly prevalent infection in human population.

Ad genome is composed of linear, non-segmented dsDNA, size between 26 and 45 Kbp; the virus can contain up to 22-40 distinct genes. Its genome is significantly larger than that of other viruses within its group, yet it is still considered a simple virus and is extremely dependent on host cell for life cycle including replication. The DNA has a terminal 55 kDa protein withineach of its 5' ends of its linear dsDNA, which serve as primers in viral replication and ensure that all of the viral genome is replicated.

Adenovirus is Key Component: Benefits and Risk Factors

Adenovirus (Ad) based cancer gene therapy is an innovative and novel form of treatment that is used as an alternative today. Gene therapy is a new approach to traditional therapies to combat severe forms of cancer. Gene delivery is used to “correct” and rebuild broken down and infected tissue within the body.

Ad is highly coveted for its ability to effectively transduce cells both dividing and non-dividing. It is also easily manipulated and has the ability to produce high titers. Infection of Ad is mediated by the binding of the fiber-knob region to the receptor of target cell, the coxsackie-Ad receptor (CAR), for most serotypes in Ad, but most commonly serotype 5. Entry and internalization of the virus is mediated by an interaction between the penton-base Arg-Gly-Asp (RGD) and cellular αvβ integrins, leading to the endocytosis of the viron through clathrin-coated pits. The virus disassembles in the endosome while the viral DNA is transported to the nucleus, resulting in expression of viral genes or transgenes. Ad DNA is not integrated in the host genome so the risk of mutation is extremely low.

Secondary receptor interactions, heparan sulfate proteoglycans (HSPGs), have also been reported. Expression of CAR is also a rate-limiting factor for infectivity with Ad5 and is highly variable in primary human tumor cells. This loss of CAR expression may be due to the progression of malignant tumors and its aggressiveness. CAR is localized within tight junctions, playing a role in cell adhesion. Ad vectors have been used for many gene therapy applications. There has been little experimental data of gene transfer with Ad, giving little evidence to significant benefits, with only Phase I and II trials to date. Many approaches to other targets and capsid modifactions are also being researched.

Ad infection enlists a wide variety of immune response both humoral and cellular, towards Ad and already infected cells. Activation of the immune system allows for a natural response to combat tumor antigens but is risky in that too high a dose will result in acute toxicity and autoimmunity. To date, side effects have been mild in Ad therapy, yet non-target side effects are consistently be monitored, both long and short term in patients.

Researchers are currently trying to increase the Ad gene transfer vectors target to tumor cells and decrease any targeting to the liver. There are a number of methods currently being solicited including: Ad fiber pseudotyping, or an alteration of virus tropism through the substitution of receptor binding-proteins with those from other serotypes, for example, the substitution of Ad5 with a knob from Ad serotype 3 (Ad3). Experiments with the Ad5/3 chimeras have showed enhanced infectivity in cancer cells without increasing gene delivery to murine liver. Adenoviruses have been isolated from a large number of different species, 100 serotypes, 57 in humans. Most people have been exposed to the adenovirus serotypes used in gene therapy, serotypes 5 and 2. Adenoviral vectors rapidly infect a broad range of human cells, yielding high levels of gene transfer compared to levels achieved with other current available vectors. Adenoviruses have low pathogenicity in humans, and cause mild symptoms. Ads used as vectors also have the capacity to hold large segments of DNA, 7.5 kil bp and transduce these transgenes in nonproliferating cells. Viral genome does not undergo rearrangement at a high rate, and inserted foreign genes are generally maintained without change through successive viral replication. Ad vectors are relatively easy to manipulate with recombinant DNA techniques.

Current Gene Therapy Studies and Results

Three regions of the viral genome can accept insertions or substitutions of DNA to generate an independent therapeutic virus vector: a region in E1, E3, and a short region between E4 and end of the genome. In the first-generation vectors, the E1 region was removed and replaced with a therapeutic transgene, prohibiting viral replication. However, viral transcription of remaining viral genes still occurred, resulting in early innate host cytokine transcription and antigen immune responses. This resulted in a reduction of the period of gene expression because of cell-mediated destruction of the transduced cells. Overall, this destruction reduced period of gene expression. Strong immune response has deemed many studies unsuccessful, with a decrease in gene expression.

Early trials studied effect of suppression of immune response through natuarally co-administration of low-dose etoposide, a cytotoxic agent and anticancer drug, but was med with limited success. Second and third adenoviral vectors have deletion in E1, E2 and E4 genes, because viral proteins encoded within these induce most of the host immune response. These new vectors exhibit decreased toxicity and result in prolonged gene expression in vivo. An important limitation in use of recombinants is the difficulty in obtaining efficient gene transfer upon second administration of virus because of antibodies. B-cells and T helpers which are part of the major histocompatibility complex (MHC)class II presentation of input viral proteins- cannot be prevented by a simple re-design of vector

There are also serious limits to adenoviral-vector gene therapy, as treatment should only be designated to cases where temporary protein expression is critical. The viral DNA does not integrate and eventually disappears, so any treatment for a chronic conditions, would need many doses and repetitions designated intervals. If only short-term therapy of the gene is required, in order to stimulate the immune system against cancer or induce apoptotic stimuli, this ad vector is suitable
Advantages to non-host- integrated genes in the chromosome are low disturbances in ceullular, genes or other processes within the body. IN pre-clinical trials, vectors have been found to be expressed in the liver, skeletal muscle, heart, brain, lung, pancreas and humor tissues. Administration of adenovirus intravenously, resulted in virus accumulation in the liver. Treatment close to reproductive organs was designated as safe, as no offspring exhbit germline transmission.

Mechanisms and Developments

Results so far have been unsuccessful. The first generation of Ad vectors were unable to replicate within cells, though could penetrate through cancer cells. A novel approach to increasing Ad vector therapy in increasing transduction of tumors is the use of replication-competent oncolytic agents, or conditionally replicative Ads (CRAds). The development of CRAds has focused on the genetic engineering of early 1, E1 genes. Major tasks of E1 proteins are to induce expression of viral genes and modifications of cellular gene expression and protein activity that favor viral replication. Two strategies that have been used to restrict virus replication to target cells and to spare normal tissue- genetic complementation-type (type 1) CRAds, like Ad524, have a mutation in the immediately early E1A or E1B adenoviral region, complemented in tumor cells but not in normal cells. In transcomplementation, the second type, CRAds, virus replication is controlled a tumor/tissue specific promoter. Ectopic liver transduction is the major predicate of Ad-vector induced toxicity.

The prototype of type 1 CRAds is dl1520 or ONYX-015. ONYX-015 contains two inactivating mutations in the gene encoding for E1B55k protein. E1B55k binds and inactivates the tumor-suppressor protein p53. The rationale is that the virus can only replicate in cells which p53 or p14ARF is mutated. CRAd does not replicate in tumor cells more than in normal cells- lack of E1B55k functions different from activation of p53. Another type-1 CRAd is through a mutation of E1A, like Ad5Δ24 or CB016, which have specific mutations in individual domains of the protein. This use of CRAd hopes to remove S phase induction by CRADs, not required in proliferating tumor cells or cervical cells transformed by human papillomavirus (HPV) viruses.

Type 2 CRAds include a tissue-specific promoter (TSP), for specific target cells. Promoter fragments with specific activity used in Type 2 CRAds are to date rare, as structure-function relationship of promoter regions has not been well characterized. TSPs have only been observed empirically, however hold strong promise. One of the most promising TSP is for ovarian cancer, secretory leukoprotease inhibitor (SLPI), a 11.7kDa serine protease inhibitor, highly expressed in different human carcinomas like breast, lung, endometrium and ovarian cancer. Gebe exoression analysis has been used to demonstrate that SLPI is 60-fold upregulated in ovarian carcinomas compared to normal ovarian epithelium. SLPI is expressed minimally in normal liver.

Recently a chimeric 5/3 fiber modified SLPI CRAd demonstrated a strong antitumor effect compared to wild-type Ad. In murine ovarian cancer, Chimeric SLPI CRAd was highly effective. The promoter of vascular endothelial growth factor (VEGF) is effective at targeting molecule for the treatment of non-small cell lung cancer (NSCLC). VEGF is also one of the most important inducers of angiogenesis, upregulated in various cancers.

Midkine is a heparin-binding growth factor identified as a retinoic acid-responsive gene; it has been reported to be over-expressed in a wide number of malignant tumors. Midkine promoter has been used successfully for vector gene delivery and CRAD targeting approaches. Promoters of CXC chemokine CXCR4 and Survivin, have optimal profile of activity and specificity in different tumor types. CXCR4 expression is upregulated in breast cancer cells but not detectablie in normal mammary epithelial cells. Survivin is a member of inhibitor of apoptosis (IAP) protein family and the expression levels of Survivigene has been shown to be clinical calue in detection of human gliomas. Genes expressing CXCR3 and Survivin play a major role in progression of metaasis in various tumors. Survivin is overexpressed in cervical intraepithelial neoplasias (CINs) and squamous cell ceinomas (SCC) of uterine cervix.

Ad based gene therapy holds the most promise for gene delivery. Although first-generation were unsuccessful, replicating Ad vectors offer hope and have been safetly and effective in clinical trials. Molecular alterations, like genetic targeting or chimeric fiber in second generation virotherapeutics in pre-clinical trials have shown improvement. Outcome of clinical trials using tropism modified CRAds will be of special interest. Pre-clinical efficacy and safety data are preliminary owing to the limitations of currently available animal models. Development of in vitro toxicity assay including precision cut human liver slices and preliminary data has demonstrated vast ability of transcriptional targeting to direct viral replication to target cells.

Gene therapy strategies combine with a multimodality treatment have the potential to cure cancer patients. Major challenges in development include effective gene therapy vectors since vectors available currently result insufficient gene transfer. Targeting of viral vectors to cellular structures with tumor cells needs further development, in order to penetrate solid tumor masses. Current generation of replication-competent Ads demonstrate superior tumor penetration and oncolysis, compared with first generation viruses. Replication competent viruses will be paired with therapeutic transgenes, like cytokines. Deterioration of solid tumors is a process, and successful gene therapy approaches will require several rounds of viral administration, whose efficacy may be inhibited by neutralizing antibodies. Strategies to reduce effect of antibodies, include alternating related viruses with different capsids or co-treatment with immunosuppressive drugs for temporary abolition of antibodies.