Current cancer treatment is based mainly on chemotherapy, radiotherapy and surgery. Despite a high rate of success when the treatment is applied at early stages, most cases of advanced disease are not curable because tumors cannot be excised by surgery or radio and chemotherapy doses that can be administered are limited by toxicity to normal cells. To alleviate this problem biotechnology strategies that seek higher selectivity and potency have been developed. Among them, gene therapy and virotherapy use viruses with a therapeutic aim against cancer. In gene therapy the virus is modified to avoid its replication and to function as a vehicle or vector of therapeutic genetic material. Conversely, virotherapy uses viruses that replicate and propagate selectively in tumor cells. In virotherapy the tumor cell dies by the cytopathic effect caused by the replication of the virus inside the cell rather than by the effect of a therapeutic gene. The preferential replication in a tumor cell is known as oncolysis. Viruses that replicate selectively in tumors are known as oncolytic viruses.
Cancer virotherapy is older than gene therapy. First reports on cancer cures with viruses date to the beginning of the past century. In 1912 De Pace obtained tumor regressions after the inoculation of rabies virus in cervical carcinomas (De Pace N. Sulla scomparsa di un enorme cranco vegetante del collo dell'utero senza cura chirurgica. Ginecologia 1912; 9:82-89). Since then, many types of viruses have been injected in tumors to treat them. There are viruses that present a natural oncotropism such as autonomous parvovirus, vesicular stomatitis virus and reovirus. Other viruses can be genetically manipulated to achieve selective replication in tumors. For example, Herpes Simplex virus (HSV) has been rendered oncotropic by deleting the ribonucleotide reductase gene, an enzymatic activity not necessary in cells ongoing active proliferation such as tumor cells. However, adenovirus, due to its low pathogenicity and high efficacy to infect tumor cells has been the most commonly used virus in virotehrapy and gene therapy of cancer.
Fifty one serotypes of adenovirus have been identified and grouped in six differentiated groups, A to F.
The human adenovirus type 5 (Ad5), which belongs to group C, consists of an icosahedral protein capsid which contains a linear DNA of 36 kilobases. In adults, Ad5 infection is often asymptomatic and causes colds and conjunctivitis in children. In general terms, Ad5 infects epithelial cells, which in a natural infection are the bronchial epithelial cells. It enters the cell by means of the interaction of the fiber, a virus protein that extends as an antenna from the twelve vertexes of the capsid, with a cellular protein involved in intercellular adhesion known as Coxsackie-Adenovirus Receptor (CAR). When the virus DNA reaches the nucleus, the transcription of early genes (E1 to E4) begins. The first genes to be expressed are those from the early 1A region (E1A). E1A binds to cellular protein pRb (retinoblastoma protein) to release the transcription factor E2F to activate the transcription of other virus genes such as E2, E3, and E4, and of cellular genes that activate the cell cycle. On the other hand, E1B binds to the transcription factor p53 to activate the cell cycle and to inhibit the apoptosis of the infected cell. E2 encodes proteins for replication of the virus. E3 encodes proteins that inhibit the antiviral immune response. E4 encodes proteins to transport virus ARN. The expression of early genes leads to the replication of the genome and, once replicated, to the activation of the major late promoter. This promoter drives the expression of an mRNA that is processed by differential splicing to give all the RNAs that encode the structural proteins that form the capsid.
As particularly relevant to the present invention, E3 proteins are described with more detail. At the early phase of the virus life cycle, before replication of the genome, E3 genes are expressed from the E3 promoter. This promoter drives the expression of a pre-mRNA which generates nine different mRNAs by splicing. In most of the serotypes of the adenovirus, namely those of the groups B, C, D and E, seven proteins (polypeptides) are synthesized from these mRNAs: E3-12.5K, E3-6.7K, E3-19K, E3-11.6K (also known as adenovirus death protein or ADP), E3-10.4K (RIDalpha), E3-14.5K (RIDbeta), and E3-14.7K (from left to right position in the genome). At the late phase the E3 promoter is repressed and the major late promoter is activated. From this promoter one pre-mRNA is synthesized which gives different mRNAs by splicing. The only E3 protein synthesized from these late mRNAs is E3-11.6K (ADP). ADP or E3-11.6K is an integral membrane protein located at the nuclear, golgi and endoplasmic reticulum membranes. It plays a role in the lysis of the infected cell. The remaining E3 proteins have functions related with the inhibition of the immune response against the infected cell. For example, E3-6.7K, RIDalpha, RIDbeta and E3-14.7K protect the cell from TNF-mediated apoptosis. E3-19K is a membrane protein that retains the major histocompatibility class 1 proteins (MHC-I) at the endoplasmic reticulum. Hence E3-19K avoids antigen presentation in the membrane of the infected cells. There are two key peptidic regions or domains to mediate this E3-19K function. One is the E3-19K MHC-I binding domain. The other one is a peptidic sequence at the carboxy-terminus end of E3-19K that retains the protein in the endoplasmic reticulum and avoids its transit to the cellular membrane. The description of these functional domains of E3-19K with mutations specific to these domains has been performed using E3-19K isolated in expression plasmids (Gabathuler R, Kvist S. The endoplasmic reticulum retention signal of the E3/19K protein of adenovirus type 2 consists of three separate amino acid segments at the carboxy terminus. J Cell Biol 1990; 111 (5 Pt 1):1803-10).
There are two important points to consider regarding the design of oncolytic adenoviruses: selectivity and potency. To achieve selectivity towards a tumor cell two strategies have been used: the deletion of virus functions that are not necessary in tumor cells and the substitution of viral promoters with tumor selective promoters. With such genetic modifications, a considerable level of selectivity has been obtained, with a replication efficiency in a tumor cell 10000-fold higher than in a normal cell. With regard to oncolytic potency several genetic modifications to increase it have been described as well. These modifications affect either the entry of the virus in the cell or the release of virus from the cell. To increase the entry step, the capsid proteins that the virus uses to infect the cell have been modified. For example, the insertion of the RGD peptide (Arginine-Glycine-Asparagine motif) in the fiber allows adenovirus to use integrins to dock in the cell and not only to internalize as it is the case with wild type adenovirus. The use of integrins as cellular receptors of the virus increases the infectivity and the oncolytic potency. Regarding the modifications that increase the release of virus from the infected cell, two have been described: the deletion of E1B-19K and the overexpression of E3-11.6K (ADP). E1B-19K is an apoptosis inhibitor homolog to Bc1-2. E1B-19K deletion increases cell death by premature apoptosis of the infected cell. This premature apoptosis often results in a lower total virus production in many infected cell lines, however it accelerates the fast release of virus and, in turn, the spread of virus in a cell culture. Accordingly the mutants that do not express E1B-19K present a large plaque phenotype compared to the wild type adenovirus in a plaque assay. Another strategy used to increase the oncolytic potency of adenovirus is the overexpression of E3-11.6K (ADP) protein. This protein plays a role in the lysis of the infected cell and ADP overexpression increases the release of the virus accumulated inside the nucleus. The phenotype of ADP-overexpressing viruses is also characterized by large plaques and the presence of more viruses in the supernatant of infected cells. ADP overexpression has been achieved by two mechanisms: 1) Eliminating the other E3 genes except ADP, or except ADP and E3-12.5K. This deletion removes other splicing sites in the pre-mRNA driven by the E3 promoter. Without the competition for these splice sites, the processing of the mRNA encoding ADP is favored. 2) Inserting the ADP gene after an strong promoter.
The present invention discloses a novel and improved mechanism to increase the release of adenovirus from the infected cell based on a mutation of E3-19K protein.