Hybrid prokaryotic-eukaryotic vectors for targeted gene delivery to brain tumours in animal models

Lead Research Organisation: Imperial College London
Department Name: Dept of Medicine

Abstract

We aim to develop a novel strategy for the treatment of human glioblastomas. These brain tumours are one of the most lethal malignancies. Despite progress in neurosurgery, radiation and chemotherapy, little progress has been made in the treatment of these tumours. Therefore, new therapeutic strategies are urgently needed.

Gene therapy is an attractive approach for cancer treatment. Glioblastomas were one of the earliest targets of cancer gene therapy, but the first clinical trials were not successful because of the inefficient delivery of therapeutic genes into the tumours. Thus, there is a need to develop new vector systems that are safe and able to provide strong expression of the therapeutic genes within the tumours. To achieve these two goals, these vectors need to be specifically targeted to the tumour tissue.

We have recently showed that combination of attributes of various viruses resulted in the generation of hybrid viral vectors for efficient and targeted systemic gene therapy. We will use this strategy to generate a novel hybrid vector for targeted delivery of therapeutic genes to glioblastoma.

These approaches could result in the derivation of a targeted vector system which would represent a major advance in the management and therapy of human glioblastoma.

Technical Summary

Human malignant gliomas are highly-aggressive central nervous system tumors. The effect of current therapies is limited, and novel therapeutic approaches are required to improve the treatment of brain tumors. Gene therapy has shown early promise and presents an interesting approach for intervention; however, in brain tumor patients, efficacy has been hindered by low tumor transduction rates.
We have reported (Hajitou et al. Cell 2006) the generation of targeted hybrid prokaryotic-eukaryotic vectors, which are chimeras between two single-stranded DNA viruses, an adeno-associated virus (AAV) and targeted bacteriophage (termed AAV phage; AAVP). In our prototype, the AAV transgene cassette is inserted in an intergenomic region of the bacteriophage (phage) genome and is packaged with the phage DNA into the capsid. In addition, the vector displays the RGD 4C ligand on the phage capsid to specifically target alpha v integrin receptors overexpressed in many tumors. The combination showed improved systemic delivery and transduction for imaging and therapy.
In this application, we propose to develop a new chimeric phage-based vector for targeted delivery of therapeutic/imaging genes to angiogenic vasculature and tumor cells in brain tumors. Specifically, we will increase vector transport through the blood-brain barrier by displaying on the bacteriophage capsid ligands for transcytosis such as transferrin. Moreover, we will combine attributes of prokaryotic and eukaryotic viruses to improve endosomal escape so as to achieve superior tumor transduction. Thus, these approaches could result in the derivation of an improved targeted vector system which would represent a major advance in the management of brain tumors and the development of gene therapy systems for patient use.

Publications

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Asavarut P (2014) The phage revolution against antibiotic resistance in The Lancet Infectious Diseases

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Chira S (2017) CRISPR/Cas9: Transcending the Reality of Genome Editing. in Molecular therapy. Nucleic acids

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Dobroff AS (2016) Towards a transcriptome-based theranostic platform for unfavorable breast cancer phenotypes. in Proceedings of the National Academy of Sciences of the United States of America

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Hajitou A (2008) A preclinical model for predicting drug response in soft-tissue sarcoma with targeted AAVP molecular imaging. in Proceedings of the National Academy of Sciences of the United States of America

 
Description BCC Small Pilot Grant
Amount £19,661 (GBP)
Organisation Breast Cancer Campaign (BCC) 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2010 
End 09/2011
 
Description Centenary Award
Amount £100,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 01/2013 
End 09/2013
 
Description Delivery of therapy to childhood CNS tumours
Amount £333,809 (GBP)
Organisation Children with Cancer UK 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2018 
End 01/2021
 
Description Drug Discovery Committee, Biotherapeutic Panel.
Amount £288,000 (GBP)
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2018 
End 06/2021
 
Description PhD student bench fees.
Amount £16,000 (GBP)
Organisation Government of Thailand 
Sector Public
Country Thailand
Start 03/2012 
End 09/2013
 
Description PhD student support
Amount £76,308 (GBP)
Funding ID P47733 
Organisation Brain Tumour Research Campaign (BTRC) 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2013 
End 05/2015
 
Description Project grant application.
Amount £219,854 (GBP)
Funding ID 2013/147 
Organisation Children with Cancer UK 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2014 
End 02/2017
 
Description Project grant application.
Amount £27,000 (GBP)
Organisation French Muscular Dystrophy Association (AFM) 
Sector Charity/Non Profit
Country France
Start 03/2011 
End 02/2012
 
Description Project grant application.
Amount £70,000 (GBP)
Organisation Brain Tumour Research Campaign (BTRC) 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2012 
End 03/2013
 
Description Proof of Concept Fund
Amount £18,000 (GBP)
Funding ID 6794 
Organisation Imperial Innovations 
Sector Private
Country United Kingdom
Start 09/2014 
End 08/2015
 
Description Supplement Funds
Amount £34,500 (GBP)
Organisation Brain Tumour Research Campaign (BTRC) 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2015 
End 07/2015
 
Description The Leverhulme Trust Fellowship
Amount £40,500 (GBP)
Organisation The Leverhulme Trust 
Sector Academic/University
Country United Kingdom
Start 04/2010 
End 12/2011
 
Title Efficacy of gene therapy by bacteriophage is improved by hybrid phage/polymer nanomaterials. 
Description The research reagent is hybrid nanomaterials of bacteriophage (phage) and cationic polymers. These phage/polymer hybrids show substantial increase of targeted gene delivery by bacteriophage, and subsequently dramatic enhancement of bacteriophage gene therapy. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact A patent application has been filed by Imperial Innovatiopns. The hybrid of phage and cationic polymers should have a notable impact on gene transfer by bacteriophage and expand the application of phage in gene transfer technology in several fields, from gene therapy to DNA vaccine delivery, both in academia and industry as well. 
 
Title Multifunctional bacteriophage nanoparticles 
Description We have designed a multifunctional filamentous bacteriophage (phage) as a new hybrid between two M13 filamentous phage fUSE5 and f88-4. This phage chimera serves i) as a carrier for a mammalian transgene cassette inserted in an intergenomic region of the bacteriophage genome for gene delivery to mammalian cells ii) as a display system of a tumour targeting ligand on the pIII minor coat protein of the fUSE5 phage and iii) as a genetic carrier for foreign functional peptides to be displayed on the pVIII major coat protein of f88-4 phage in order to operate as a nanoparticle (nanotube) decorated by hundreds of anticancer peptides. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact The novel multifunctional phage will be applied as vehicle to target cell surface receptors in activated endothelium-derived cells or tumour cells in mouse tumour models. Target specificity will be based on tumour vascular homing ligands displayed on the phage coat. The cargo will be a packaged gene incorporated into the phage genome for gene delivery applications, or imaging agents or cytotoxic peptides displayed at high density onto the phage coat. This strategy is likely to lead to clinical applications of phage in nanomedicine related to therapy and imaging and would represent a major advance in the management of cancer and other human diseases. 
 
Title Phage pseudotyped Adeno-associated virus (P-AAV) 
Description A new simple way of gene delivery by a bacteriophage vector named phagemid-AAV 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? No  
Impact This new phage vector has substantial impacted targeted gene delivery and is the most advanced phage vector in targeted gene therapy. 
 
Description Association of histone modification and DNA methylation status with efficacy of glioblastoma gene therapy by bacteriophage. 
Organisation Imperial College London
Department Division of Experimental Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution My research technician, funded by my MRC award, used inhibitors of histone deacetylation and DNA methylation to show that modulation of the histones and DNA methylation status enhance bacteriophage gene therapy against glioblastoma.
Collaborator Contribution My partners provided us with inhibitors of histone deacetylation and DNA methylation as well as guidance for the work, given their expertise in the epigenetic field.
Impact PMID: 24153059
Start Year 2011
 
Description Design and generation of novel M13 filamentous bacteriophage vectors with double-stranded DNA. 
Organisation University College London
Country United Kingdom 
Sector Academic/University 
PI Contribution We have used a panel of drugs, such as Genestein, known for their ability to convert single-stranded to double-stranded DNA, and showed that they have the ability to increase gene transfer efficacy to gliobalstoma cells by our single-stranded bacteriophage vector.
Collaborator Contribution Our partners will aim to establish that this enhanced gene delivery by bacteriophage, in glioblastoma cells, is the result of efficient conversion to double-stranded DNA. They will also provide us with self-complementary AAV transgene cassettes to insert into our bacteriophage vector in order to generate double stranded-DNA in transduced cells and subsequently increase gene expression by bacteriophage in cancer cells.
Impact A patent application is being prepared.
Start Year 2013
 
Description Phage therapy against medulloblastoma 
Organisation University of Portsmouth
Department School of Pharmacy and Biomedical Sciences Portsmouth
Country United Kingdom 
Sector Academic/University 
PI Contribution We have generated multifunctional phage particles for the simultaneous targeting of molecular targets in medulloblastoma.
Collaborator Contribution They provide a panel of human medulloblastoma cell lines.
Impact Collaboration with the University of Portsmouth.
Start Year 2011
 
Description Phage-guided CAR T cell immunotherapy against CD19 antigene in cancer 
Organisation Advent Life Science
Country Unknown 
Sector Private 
PI Contribution We will construct bacteriophage vectors to deliver CD19 gene expression to tumours in vivo following intravenous administration.
Collaborator Contribution Our partners will provide CAR T cells displaying an antibody against CD19 on their cell surface.
Impact The collaboration is just starting.
Start Year 2016
 
Description Phage-guided cancer DNA and peptide vaccines 
Organisation University of Oxford
Department Jenner Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution My team will has constructed tumour targeted bacteriophage vectors for intravenous delivery of foreign antigens to tumours in preclinical models.
Collaborator Contribution My partners have provided us with the foreign antigens to use. They will also perform in vitro tests in their lab.
Impact The experimental work has started in October 2015.
Start Year 2015
 
Description Phage/polymer hybrid nanomaterials 
Organisation Imperial College London
Department Department of Chemical Engineering
Country United Kingdom 
Sector Academic/University 
PI Contribution My PhD student designed hybrids of targeted bacteriophage and cationic polymers, and subsequently converted the negative charge of bacteriophage into a positively charged surface. Then he showed that the new positively-charged phage capsid acquired strong attachment to the negatively charged membranes of cancer cells, resulting in dramatic increase of gene delivery and targeted tumour cell killing by bacteriophage.
Collaborator Contribution Our partners carried out an extensive physicochemical characterisation of the new phage/polymer hybrid nanomaterials.
Impact 1- The work has been published in Moelcualr Therapy- Nucleic Acids, 2014. Title: Hybrid nanomaterial complexes for advanced phage-guided gene delivery Authors: Teerapong Yata, Koon-Yang Lee, Tararaj Dharakul, Sirirurg Songsivilai, Alexander Bismarck, Paul Mintz and Amin Hajitou. 2- A patent application was also filed by Imperial Innovations.
Start Year 2012
 
Description Targeted T-cell activation by tumour-associated neo-vasculature 
Organisation King's College London
Department School of Medicine KCL
Country United Kingdom 
Sector Academic/University 
PI Contribution On our side we provide the vascular-targeted bacteriophage vector in order to express on the surface of tumour blood vessels the necessary ligands that guide T-lymphocytes to the tumour vasculature for a targeted cancer immunotherapy.
Collaborator Contribution My collaborator has provide research material needed to initiate the project.
Impact We are in the process of generating preliminary data to demonstrate the feasibility of combining our technology with that of our collaborator. This will allow us to apply for funding in order to pursue this very promising combination of the two technologies.
Start Year 2010
 
Description Targeted combination therapy of intracranial human glial tumours in mice. 
Organisation Imperial College London
Department Centre for Clinical Translation
Country United Kingdom 
Sector Academic/University 
PI Contribution To grow human glioblastoma from cancer patients in the brain of mice and assess the effects of targeted gene therapy in combination with other therapies e.g. chemotherapy.
Collaborator Contribution To grow human glioblastoma cells from cancer patients and provide them to us for implantation in mice.
Impact We have published a collaborative paper: Przystal, J.M., Umukoro, E., Stoneham, C.A., Yata, T., O'Neill, K., Syed, N. and Hajitou A. "Proteasome inhibition in cancer is associated with enhanced tumor targeting by the adeno-associated virus/phage" Mol. Oncol. (2012) Aug 21. [Epub ahead of print].
Start Year 2010
 
Description Targeted systemic immune gene therapy against paediatric diffuse intrinsic glioma (DIPG) 
Organisation Hospital Sant Joan de Deu
Country Spain 
Sector Hospitals 
PI Contribution Our part has been to design and develop phage vectors carrying cytokine genes with ability to destroy human DIPG in a selective way without harming the healthy cells.
Collaborator Contribution Our collaborators have expertise in generating preclinical models of human DIPG in immune deficient mice and rats.
Impact We have developed a phage vector carrying the cytokine TRAIL that destroys human DIPG in vitro in a selective manner without affecting the healthy human tissues.
Start Year 2018
 
Description Use of bacteriophage as a delivery system in DNA vaccine applications. 
Organisation Government of Thailand
Country Thailand 
Sector Public 
PI Contribution We will supervise in my lab a PhD student funded by the Government of Thailand.
Collaborator Contribution The government of Thailand will fund a Thai PhD student who will work on the project and study the PhD under my supervision at Imperial College London.
Impact This collaboration has just started
Start Year 2014
 
Description Vascular-targeted molecular imaging and therapy of breast cancer 
Organisation Government of Thailand
Department National Science and Technology Development Agency (NSTDA)
Country Thailand 
Sector Public 
PI Contribution Our contribution is to use single chain antibodies displayed in the context of bacteriophage capsid in order to systemically target imaging and therapeutic transgenes to the angiogenic vscaulature of breast cancer in mice
Collaborator Contribution Provides a studentship support (tuition fees and living expenses) for a student from Thailand to do the PhD at Imperial College London under my supervision.
Impact The work is progressing very well and a publication is being submittded..
Start Year 2010
 
Title BACTERIOPHAGE 
Description The invention provides a recombinant targeted bacteriophage for expressing a transgene in a target cell transduced with the bacteriophage. The bacteriophage comprises a first nucleic acid sequence encoding a pill capsid minor coat protein that is configured to display a cell-targeting ligand for enabling delivery of the bacteriophage to a target cell, a second nucleic acid sequence encoding at least one pVIII capsid major coat protein that is configured to display a foreign peptide thereon, and a transgene which encodes a protein which exerts a biological effect on the target cell. 
IP Reference WO2014184528 
Protection Patent application published
Year Protection Granted 2013
Licensed No
Impact The discovery has attracted further funding, and collaborations with industrial partners.
 
Title BACTERIOPHAGE 
Description The invention provides a targeted bacteriophage-polymer complex comprising a recombinant targeted-bacteriophage and a cationic polymer. The complex has a net positive charge. The invention provides methods of preparing bacteriophages and complexes thereof, and to their uses for the delivery of transgenes in a variety of gene therapy applications. 
IP Reference WO2014184529 
Protection Patent application published
Year Protection Granted 2013
Licensed No
Impact The discovery has attracted further funding and collaborations with Industry.
 
Title Hybrid multifunctional filamentous bacteriophage nanocarrier 
Description The product: is a multifunctional filamentous phage as a new hybrid between two M13 filamentous phage. The product can be used i) as a carrier for mammalian transgene cassettes inserted in an intergenomic region of the bacteriophage for gene delivery to mammalian cells, ii) as a display system of a tumour targeting ligand, and iii) as a carrier for therapeutic peptides. Current stage: The product has been successfully tested in vitro on tumour cell lines. Next we will assess the efficacy of the product in tumour-bearing mice in vivo. Principle source of funding for this development: Medical Research Council. An international patent application has been filed by Imperial Innovations. 
Type Therapeutic Intervention - Drug
Current Stage Of Development Initial development
Year Development Stage Completed 2010
Development Status Actively seeking support
Impact Wide biotechnological and clinical applications of phage particles and a breakthrough for phage applications in nanomedicine. 
 
Title Phage/polymer hybrid nanoparticles 
Description Bacteriophage (phage), viruses that infect bacteria only, have shown promise for targeted gene transfer applications. Unfortunately, only minor progress has been made in improving their potential and enabling them to overcome mammalian cellular barriers. We hypothesized that one of the limitations of phage is its surface negative charge, which could hinder its access to negatively charged eukaryotic cell membranes and subsequent binding to the target cell receptor. Therefore, we generated a novel hybrid system consisting of two classes of nanomaterial systems, cationic polymers and the M13 bacteriophage virus particles genetically engineered to display a tumor targeting ligand and to carry a transgene cassette. We show that complexation of phage with cationic polymers generates positively charged phage which show enhanced cell surface attachment and improved transgene expression while retaining cell type specificity. Moreover, phage/polymer complexes carrying a therapeutic gene achieve greater targeted cancer cell killing than phage alone. An international patent application has been filed by Imperial Innovations. 
Type Therapeutic Intervention - Cellular and gene therapies
Current Stage Of Development Initial development
Year Development Stage Completed 2013
Development Status Actively seeking support
Impact This new class of hybrid of phage/polymer nanomaterial platform can advance targeted gene delivery applications in general, and gene transfer by bacteriophage in particular.