Potential Applications of Adenovirus-Mediated Gene Delivery in Gene and Cell-Based Therapies

There has been a long-lasting interest in using viral vectors, especially adenoviral vectors, to deliver therapeutic genes for the past two decades. Among all currently available viral vectors, adenovirus is the most efficient gene delivery system in a broad range of cell and tissue types. In fact, among over 2000 gene therapy clinical trials approved worldwide since 1989, a significant portion of the trials have utilized adenoviral vectors. It is expected that the continued improvements in adenoviral vectors should provide great opportunities for cell and gene therapies to live up to its enormous potential in personalized medicine.

The Characteristics of Adenovirus

The adenoviral genomes are linear, non-segmented double-stranded DNA with sizes ranging from 26 kb to 45 kb in length, depending on the serotype. The genome of the commonly-used human adenovirus type 5 (Ad5) is approximately 36 kb. The DNA is flanked on both ends by hair-pin-like, inverted terminal repeats (ITR), which serve a variety of purposes. In addition to ITRs, another genetic element of the adenovirus is the packaging signal, which is located on the left arm of the genome and is required for proper viral transcript packaging. Viral transcripts are classified as either early or late. The four early transcriptional units, E1, E2, E3, and E4, are responsible for expressing non-structural proteins. These proteins have regulatory functions and are involved in viral DNA replication. The late proteins encode for structural components of the Ad virion.

Fig. 1 The genome structure and major transcript units of human Ad5.

Approaches to The Efficient Productions of Recombinant Adenoviral Vectors

Although the use of recombinant adenoviruses has been widespread, the initial generation of a given adenoviral vector (mostly the first generation AdVs) remains a technically challenging and time-consuming process. There are four common methods used to produce adenoviral vectors:

The traditional method – recombination in HEK-293 cells. The gene of interest (GOI) is first cloned into a shuttle vector, which contains 5'-ITR, packaging signal and homologous regions to adenoviral genome. Adenoviruses are generated in HEK-293 cells through recombination between shuttle vector and adenoviral backbone vector, which is unable to produce virus by its self.
Cre/LoxP-mediated recombination. The GOI is cloned into a shuttle vector that contains LoxP site(s). Cre recombinase-mediated recombination occurs with a LoxP-containing adenoviral backbone vector in vitro or 293-Cre cells, leading to the generation of recombinase adenoviruses.
The AdEasy system. The GOI is subcloned into a shuttle vector that contains 5'-ITR and packaging signal, as well as a kanamycin-containing bacterial replication unit flanked with homologous arms. Recombinant adenoviral plasmids are generated through homologous recombination between the linearized shuttle vector and ampicillin-resistant adenoviral backbone vector, such as pAdEasy1, in the bacterial strain BJ5183 cells under kanamycin selection. The resultant adenoviral plasmids are linearized and used for adenovirus production in HEK-293 cells.
The use of helper adenovirus for the production of HC-AdVs (or HD-AdVs, or Gutless AdVs). The GOI is cloned into a transfer vector that contains both ITRs and packaging signal only. Adenoviruses are generated with a helper adenovirus, which will not be packaged due to the deletion of packaging signal in the modified HEK-293 cells, usually through Cre/LoxP or FLP/FRT excision system.

Fig. 2 Four commonly-used methods to generate and produce adenovirus vectors for gene delivery.

Adenoviral Vectors in Gene and Cell-Based Therapies

Adenovirus-mediated gene therapy for monogenic diseases

The first in vivo application of AdVs for genetic therapy in humans was reported in 1992 with the successful transfer and expression of alpha-1 antitrypsin (A1AT) cDNA within hepatocytes of a patient with alpha-1 antitrypsin deficiency. With each subsequent administration, investigators came to recognize that a response by the patients' humoral and cellular immunity to adenoviral vectors was responsible for this transient, 1-2 week expression of AdV transgenes, while also leading to adverse immunogenic events in other patients. Much of the research focus in that time then shifted toward developing less immunogenic viral vectors. Hence, the utility of AAV and lentiviral vectors emerged. Despite these initial setbacks, AdVs have returned to the spotlight of human gene therapy in recent years. The potent immunogenic properties that once undermined the use of AdVs in sustained treatment of genetic deficiencies have since been leveraged to purposefully evoke host immunity, thereby highlighting the adenovirus as an ideal vaccine carrier and antitumoral agent.

Adenoviral vectors as anticancer agents

There are three main therapeutic categories to which AdVs are applied as anticancer therapies. In the first, replication-defective (RD) AdVs are used for their immunogenic properties to deliver immune-related genes/epitopes directly to tumor cells in order to attract and induce a local antitumoral immune response. In the second category of strategies, replication-competent (RC) AdVs may be used to preferentially replicate within cancer cells and achieve oncolysis by executing the natural lytic life cycle of the virus in cancer cells. Lastly, either RD or RC AdVs may be used to deliver and/or overexpress tumor-suppressor genes or cytotoxic/suicide genes within cancerous cells to directly induce an intrinsic cytotoxic cascade, cause cell cycle arrest, or trigger apoptosis.

Fig. 3 The anticancer effect of oncolytic adenoviruses (CRAdVs).

Adenoviral vectors for vaccine development

Adenoviral vectors have been incorporated into the development of vaccines against tuberculosis, HIV, Ebola, malaria, and influenza. Different from most traditional vaccines, immunization techniques using recombinant viral vectors trigger both a robust cytotoxic T-cell response in addition to a humoral response. This cytotoxic T-cell response is far more efficient in destroying virus-infected cells, intracellular pathogens, and cancerous cells – while also expanding protective immunity against strains of similar pathogens that express homologous epitopes.

Adenovirus-mediated gene delivery in regenerative medicine

AdV-mediated gene delivery has also been used in numerous preclinical studies involved other aspects of regenerative medicine and tissue engineering. The study demonstrated that AdV-mediated expression of Sox9 significantly improved the healing of intervertebral degenerative discs in a rabbit model. Adenoviral vectors were shown to deliver transgenes effectively into the flexor tendon of rabbit injury model.

Reference

Lee, C. S.; et al. Adenovirus-mediated gene delivery: potential applications for gene and cell-based therapies in the new era of personalized medicine. Genes & diseases. 2017, 4(2): 43-63.

* For research use only. Not intended for any clinical use.

Quick Inquiry

Potential Applications of Adenovirus-Mediated Gene Delivery in Gene and Cell-Based Therapies

The Characteristics of Adenovirus

Approaches to The Efficient Productions of Recombinant Adenoviral Vectors

Adenoviral Vectors in Gene and Cell-Based Therapies

Related Products and Services at Creative Biogene