Characterisation of a novel phage-guided gene delivery vector and investigation of its gene therapy efficacy against metastatic cancer.

The majority of cancer patients die because of metastases which are established after cancer cells leave their initial site or primary tumour in order to spread and form secondary tumours or metastases in other tissues. Failure of conventional therapies (surgery, radiotherapy and chemotherapy) to treat metastases is associated with the high number of metastases located in various tissues, requiring systemic treatment to reach all these metastases. Conventional systemic chemotherapeutic agents have been used but often ineffective, mostly because they are not selective, resulting in their accumulation in healthy tissues, which comes with sever side effects limiting the chemotherapeutic dose to be injected to patients.
Gene therapy, or therapy using genes, is a promising treatment approach against metastatic cancer. Gene therapy requires a vector (vehicle or delivery system) to carry the therapeutic gene and achieve its delivery at the tumour site. Human viruses have mostly been used to design vectors of gene therapy because they can enter human cells and deliver therapeutic genes as part of their natural infection process. Gene therapy for cancer has been attempted for more than 26 years. However, like chemotherapy, gene therapy has faced a major challenge that has also been inefficacy after systemic administration, limiting its effectiveness against metastases since real clinical benefit against metastatic cancer can only happen with systemic gene therapy. These challenges are associated with vectors of gene therapy, since the vectors accumulate in various healthy tissues and get neutralized by the body immune response against these viral vectors. Our previous work shows that the harmless and non-pathogenic bacteriophage or phage, viruses that infect bacteria only, can deliver genes to human cells if they are engineered to display ligands on their capsid. These ligands allow the bacteriophage to bind to receptors on human cells resulting in their entry into the cells and expression of the therapeutic genes. If the target receptor is specific in cancer, then the phage vector becomes targeted after systemic administration to deliver therapeutic genes to cancer while sparing the healthy tissues. Our first generation of these phage vectors showed safety and anti-tumour efficacy in preclinical models of cancer after intravenous administration. We have spent the last 10 years to improve these vectors and generate systems that can overcome their limitations since bacteriophages have evolved to infect bacteria only with no developed strategies to express genes in human cells. Indeed efficacy of gene therapy depends on the ability of vectors to express genes at therapeutic levels in tumours. Importantly, very recently our efforts have yielded a bacteriophage vector that could make a breakthrough in systemic gene therapy of metastatic cancer. It is important to undertake this work because the vector shows ability to overcome major limitations that cancer gene therapy has faced and could bring to fruition the promise of gene therapy to save the lives of patients with deadly metastatic cancers.

Successful treatment of metastatic cancer is refractory to strategies employed to treat confined, primary lesions and thus must be addressed by systemic delivery of anti-cancer agents. Conventional systemic chemotherapy is often ineffective and comes with severe dose-limiting toxicities. Improving specificity of action appears to be key goal in the development of novel treatments against metastases. Gene therapy has been attempted against cancer for decades; but has been significantly hindered by lack of tumour-selective vectors through systemic administration and by problems with repeated delivery. We have introduced a unique prokaryotic viral-based approach of intravenous gene delivery to target tumours specifically by using the harmless filamentous M13 bacteriophage (phage), a virus that infects bacteria only. In this vector, named AAV/phage (AAVP), the M13 phage capsid delivers AAV vector genomes and was engineered to display the RGD4C ligand that binds the alpha-V/Beta-3 integrin receptor, overexpressed on tumour cells and supporting vasculature in most cancers, but barely detectable on healthy tissues. To date, we and collaborators have established that these vectors target various preclinical models of human cancer. Additionally, the vector showed efficacy in pet dogs with natural cancers. Repeated administrations proved anti-tumour efficacy in immunocompetent mice despite IgGs against the phage capsid. We have improved the technology and generated a vector with escape ability from the endo-lysosome degradative pathway. Importantly, we have further refined the hybrid vector by eliminating the phage genome, which decreased the vector size and generated a Phage-pseudotyped AAV (Ps-AAV) with large cloning capacity and improved gene delivery. We propose to investigate cellular entry and intracellular trafficking of this new hybrid Ps-AAV. Next we will investigate the vector in repeated systemic gene therapy dosing against metastatic cancer in preclinical models.

Patients with metastatic tumours should benefit from this targeted systemic gene therapy approach, as most cancer patients die because of metastases. Indeed, our gene therapy approach is delivered intravenously, a clinically non-invasive route, applicable both in localized and metastatic tumours. Moreover, our vector can be administered repeatedly, which is crucial to achieve and sustain a therapeutic response against metastases. Patients with secondary brain metastases, which are deadly with very disappointing prognosis, should also benefit given the natural ability of the vector to cross the blood-brain barrier and target brain tumours as we recently reported in (Przystal et al. EMBO Molecular Medicine, 2019). Outcomes from the proposed studies could represent a novel form of cancer therapy which may find broad applications.
It is noteworthy to mention that phage-guided gene therapy can rapidly enter clinical trials in cancer patients as phage has already been safely used for clinical applications in adult and children over many years, primarily to treat bacterial infections. Phage vectors are highly amenable to production to Good Manufacture Process (GMP) standard, both in EU and the UK. Outcome from our proposed research should yield a cost-effective form of cancer treatment and generate an Economical Impact. Indeed, phage vectors are economical since production and purification processes of phage vectors occur in prokaryotic hosts, which are compatible with industrial-scale reactor and separation systems. Also, phage vectors are safe and of little concern when compared to mammalian viruses, as they cannot infect eukaryotic cells. Finally, pharmaceutical companies have expressed continuous interest in our vector; we will enter into negotiations and collaboration with them for the best way forward to develop our therapeutic approach for clinical use.
As stated in the Academic Beneficiaries, future research outcome will continue to advance the acknowledge in phage-mediated Nucleic Acid delivery and improve gene delivery technologies. The outcome should also contribute to the training of the next generation of scientists and group leaders in this field and gene therapy field in general.
We will also identify potential users of the scientific and technical outcomes of our research outside the academic research community. Imperial Innovations and the office of Corporate and Entrepreneurship for business interactions, at Imperial College, will support in this area in terms of advice and funding. Pharmaceutical companies (e.g. Pfizer, Eli Lilly, PhageNova Bio, Freeline Therapeutics) have expressed their interest in Phage-pseudotyped AAV (Ps-AAV). Spin-out investment will certainly be under consideration. It is noteworthy to mention that the first generation of hybrid AAV/Phage vector (AAVP), had generated patents with the University of Texas, in which Hajitou is an inventor. Then, a spin-out company was founded and named AAVP Biosystems after the vector then the company changed its name to PhageNova Bio; the company has licenced the patents. I have collaborated with PhageNova Bio to promote vector access to clinical trials. We have successfully completed a safety and efficacy study in larger animals (pet dogs with spontaneous cancers). Phase-1 trials in cancer patients should start in 2020.