Oncolytic Adenovirus

Oncolytic adenoviruses (Ads) have emerged as a promising therapeutic tool to provide a versatile system for gene expression studies and therapeutic applications. With the increasing experience and knowledge of the Ad system, coupled with the OncoVirapy™ platform, Creative Biolabs commits to developing efficacious oncolytic adenovirus for customers and enabling wider applications of adenovirus in oncolytic virotherapy of cancers.

Brief Introduction of Ads

Ad, belonging to Adenoviridae family, is a non-enveloped, double-stranded DNA virus with an icosahedral capsid encompassing a linear duplex genome of ~36 kb. Its genome can be divided into early genes (E1A, E1B, E2, E3, and E4) and late genes (L1-L5). Early genes encode corresponding proteins that are expressed early during viral replication whereas late genes are expressed after the commencement of viral replication and encode for capsid proteins. Conveniently, the virion structure of Ads allows the insertion of complementary DNA (cDNA) elements for expression of foreign transgenes to impact their tropism, safety, tumor lysis and so on. The first recombinant adenovirus (H101, one E1B mutant adenovirus) for head and neck tumors’ treatment was approved in November 2006 in China.

Fig.1 The scheme of the wild type serotype 5 adenovirus genome. (Abudoureyimu, 2019)

Human Adenovirus Classification and Tropism

Human Ads comprising more than 60 different serotypes were historically classified into species A-G based on their DNA homology, hemagglutination, oncogenic and neutralization properties. The serotypes of Ad2 and Ad5, both belonging to group C, are the most widely studied and most commonly used vectors for gene therapy. Ads are capable of infecting a wide variety of vertebrate hosts via aerosol droplets in the respiratory, urinary or gastrointestinal tract, utilizing multiple cellular entry receptors in a serotype-dependent manner, such as human coxsackie and adenovirus receptor (hCAR), Desmoglein-2 (DSG-2), CD46, CD80, CD86, etc.

Table.1 Human adenovirus classification, tropism and receptors.

Featured Services and Construction Workflow

We provide a comprehensive range of adenovirus services based on our advanced OncoVirapy™ platform.

• GOI cloning (e.g., antibody, bispecific antibody, cytokine, chemokine, shRNA or gRNA) and confirmation through restriction mapping and sequencing verification
• Functional validation of transgenes
• Expression cassette design with the most appropriate promoter to guarantee the high-level expression
• Viral genes engineering (E1, E3, E4 region)
• Capsid (hexon, fiber) modification to alter the Ad tropism
• Hybrid adenovirus construction
• Ad amplification, purification, titering services
• Ad whole genome sequencing (WGS) service

With years of experience and continuous optimization, Creative Biolabs has developed a systematic workflow for oncolytic Ad construction as follows:

Fig.2 Workflow of oncolytic adenovirus construction at Creative Biolabs.

Genetic Modifications of Oncolytic Adenovirus Vectors

• The Ad genome is well-characterized, compact and largely permissive for a plethora of genetic modifications. Therefore, Ads allow the insertion of different foreign expression cassettes to generate recombinant adenovirus vectors, including immunostimulatory transgenes (e.g., cytokines and chemokines), non-immunostimulatory transgenes (e.g., tagging proteins such as luciferase, green fluorescent protein [GFP], or prodrug converting enzymes, and ion channels), and cDNA coding for full-length antibodies (e.g., anti-CTLA4 and anti-HER2).
• As important therapeutic gene transfer vehicles, adenovirus vectors can be engineered to possess different properties and functions. The early genes (often E1 and/or E3) are deleted and replaced with a transgene of interest. The inserted genes are typically controlled by a constitutive promoter element, such as the cytomegalovirus (CMV) or Rous sarcoma virus (RSV) promoter, which allows transgene expression in any transduced cell. Tumor-specific promoters, such as human telomerase reverse transcriptase, carcinoembryonic antigen, cyclooxygenase, and E2F, have been used in oncolytic adenovirus constructs to achieve tumor specificity replication. Additionally, fiber modification is commonly used to direct oncolytic adenovirus tumor tropism to facilitate adenovirus transduction.

Fig.3 Insertion and deletions commonly used to design oncolytic adenoviruses. (Alemany, 2016)

Cases of Ad Genetic Modifications

1. ONYX-015, the E1b-55k region was deleted which is capable of replication in p53-deficient human tumor cells.
2. H101, deleting the entire E1B gene and a 78.3-85.8 nm gene segment in the E3 region, which is responsible for the code of the adenovirus death protein.
3. Ad5-Δ24RGD carries a 24-bp deletion in the E1A region that is responsible for binding Rb protein and RGD motif insertion into the fiber.
4. ZD55 deleted the E1B 55-kD gene and armed with a foreign gene.
5. ICOVIR5, the oncolytic adenovirus in which the endogenous E1A promoter has been replaced by the human E2F-1 promoter insulated with the DM-1.
6. Gendicine is a recombinant human Ad5 in which the E1 region is replaced by a human wild-type p53 expression cassette, driven by a Rous sarcoma virus (RSV) promoter with a bovine growth hormone (BGH) poly(A) tail.

Fig.4 Examples of oncolytic adenovirus genetic modifications. (Abudoureyimu, 2019)

Oncolytic adenovirus is continuously being assessed for effective and safe future therapeutics. With the development of modern genetic engineering techniques, novel strategies have been discovered to optimize the construction of oncolytic adenovirus, to reduce their clinical toxicity, to construct efficient OV delivery platforms, and to increase the efficacy of OVs, with the aim of achieving the greatest therapeutic benefit. Creative Biolabs offers a broad range of custom adenovirus cloning and construction services at a reasonable cost and with quick turnaround time. Besides, we also provide in vitro and in vivo validation services for engineered oncolytic Ads to facilitate your whole project.

References

1. Abudoureyimu, M.; et al. Oncolytic adenovirus-A nova for Gene-targeted oncolytic viral therapy in HCC. Frontiers in oncology. 2019, 9: 1182.
2. Alemany, R. Molecular design of oncolytic adenoviruses. Adenoviral Vectors for Gene Therapy. Academic Press. 2016: 319-334.

Please feel free to contact us for a quote and further discussion with our scientists.