Viral vectors in clinical gene...
Transcript of Viral vectors in clinical gene...
Prof. Józef Dulak, PhD, DScDepartment of Medical Biotechnology
Faculty of Biochemistry, Biophysics and BiotechnologyRoom 3.025/3.07 Phone 664-63-75
Email: [email protected]
30 November 2015
Viral vectorsin clinical gene therapies
(2)
Exam – planned on 1st February (Monday), room D107, 1 pm
Immuno-gene therapychemoprotection
Tumor suppressor (p53)
Suicide gene therapy
Types of cancer gene therapy
Cancer gene therapy
1. Direct attack on tumor cellsa) transfer of tumor suppressor gene – p53 b) inhibition of oncogenes
- antisense therapy- ribozymes- microRNAs
c) suicide genesd) oncolytic viruses (replication-competent viruses)
2. Harnessing immune response to tumor antigens3. Chemoprotection4. Anti-angiogenic therapy
p53 and gene therapy of cancer
p53-mediated tumor suppressor network
Tanazawa et al., Exp Opinion Biol Ther 13 (11): 2013
About 50% of cancers have mutated p53
p53 and cancer development
TP53
Cancer cell
Adenoviral vectorWith correct p53 gene
expression of p53 incancer cell
apoptosis
Adenoviral delivery of p53 gene
First officially registered therapeutic nucleic acid
Gendicine (SiBiono GeneTech, Chiny)Adenoviral vector with a correct p53 gene
Registered on 16. X. 2003, after 5 years of clinical trials
Efficient in patients with head and neck cancers
Claimed to be 3 x more efficient than radiotherapy alone
ADVEXIN®
p53 tumor suppressor therapy developed in USAADVEXIN therapy combines the p53 tumor suppressor with a non-replicating, non-integrating adenoviral delivery system we have developed and extensively tested. Introgen's clinical trial
strategy for ADVEXIN was to test it in a variety of life-threatening cancers for which there are no effective treatments. Introgen was seeking to register ADVEXIN for the treatment of head and neck cancer and Li-Fraumeni Syndrome. Additional late stage clinical trials in breast and lung
cancers will enable Introgen to add follow-on indications.
FDA designated Fast Track Drug Product Development program
FDA and EMEA designated Orphan Drug status for ADVEXIN® in head and neck cancer.
ADVEXIN® therapy well tolerated and clinically active.
In 2008, Advexin finally was not registered by FDA…
There are doubts on the real effectiveness of Gendicine
When Bad Gene Transfer Is Good...
Cancer gene therapy
1. Direct attack on tumor cellsa) transfer of tumor suppressor gene – p53 b) inhibition of oncogenes
- antisense therapy- ribozymes- microRNAs
c) suicide genesd) oncolytic viruses (replication-competent viruses)
2. Harnessing immune response to tumor antigens3. Chemoprotection4. Anti-angiogenic therapy
Suicide gene therapy –pro-drug activation
The objective of pro-drug activation therapy is to expressan activating enzyme within the tumor, which will then
activate a systemically delivered, inactive pro-drugat the target site only
Gene-directed enzyme prodrug therapy
Bystander effect(efekt sąsiedztwa)
Suicide gene therapy –pro-drug activation
Enzyme-prodrug combination for suicide gene therapy
Enzyme Prodrug Product Mechanism
HSV-tk ganciclovir ganciclovir triphosphate blocks DNA synthesisacyclovir
valacylcovir
cytosine deaminase 5-fluorocytosine 5-fluorouracil (5-FU) blocks DNA and RNA synthesis (pyrimidine antagonist)
cytochrome P450 cyclophosphamide phosphoramide mustard DNA alkylating agent;blocks DNA synthesis
Herpes virus thymidine kinase-based suicide gene therapy
Glioblastoma multiforme - Infiltrative; rapid-growing; occurs: most frequently in mid-aged; apt to involve both cerebral hemispheres via the corpus callosum; Average Survival: 1 year
Glioblastoma multiforme
Gene therapy for glioblastoma multiforme
- surgical resection of tumor followed by radiotherapy – all patients
- delivery of AdvHSV-tk (3 x 10(10) pfu) by local injection into the woundbed after tumor resection, followed by intravenous ganciclovir5 mg/kg twice daily for 14 days - starting 5 days after surgery
- Cell division is a key characteristics of cancer, and the normal brain cells are not dividing, hence tumor cells that try to divide around the site of the removalof the original tumor are targeted for destruction
University of Eastern Finland, Kuopio
AdvHSV-tk treatment produced a clinically and statistically significant increase in mean survival from 39.0 +/- 19.7 (SD) to 70.6 +/- 52.9 weeks (P = 0.0095, log-rank regression vs. randomized controls). The median survival time increased from 37.7 to 62.4 weeks.
Mol Ther. 2004 Nov;10(5):967-72
Gene therapy for glioblastoma multiforme
Mol Ther. 2004 Nov;10(5):967-72
Cerepro™ has been granted Orphan Drug Status by the European Committee for Orphan Medicinal Products and by the Office of Orphan Products Development, FDA
1. 1998 – establishing the dose and method of administration using a marker gene
2. 2000 – results of the second, open label efficacy and safety study showed thatCerepro doubled mean survival time and was well tolerated
3. 2004 – results of the third study presented and published (Molecular Therapy)
4. ASPECT study – a phase III, randomised, controlled, multicentre evaluationthat recruited 250 patients
Cerepro – sitimagene ceradenovec
ASPECT – suicide gene therapy trial of glioblastoma multiforme
Lancet Oncol; August 2013
Standard treatment: surgery followed by chemotherapy (temozolomide)
Lancet OncolAugust 2013
ASPECT trial –Kaplan-Meier estimates of time to death or reintervention
December 2009 - the European Medicines Agency hasnot agreed to market Cerepro…
Ad-HSC-Tk is tested as therapy in various cancer
There is a hope that new methods of delivery might provide better therapeutic outcome
Cancer gene therapy
1. Direct attack on tumor cellsa) transfer of tumor suppressor gene – p53 b) inhibition of oncogenes
- antisense therapy- ribozymes- microRNAs
c) suicide genesd) oncolytic viruses (replication-competent viruses)
2. Chemoprotection3. Harnessing immune response to tumor antigens4. Anti-angiogenic therapy
JCI, September 2014
1. To date, the most effective chemotherapies to treat GBM are alkylating agents, such as temozolomide (TMZ).
2. However, despite TMZ potency, median survival in all GBM patients remain less than 2 yearsfrom diagnosis (21 months).
3. Moreover, approx. 50% of GMB tumors overexpress methylguanine methyltransferas((MGMThi), indicated by the hypomethylation of the promoter controlling MGMT expression.
4. MGMThi tumors are resistant to TMZ due to the ability of the catalytic MGMT protein to repairTMZ-induced cytotoxic DNA-damage, conferring poor median survival time (12.6 months).
5. O6BG (O6-benzylguanine) is a small nucleoside inhibitor which effetively depletes MGMT activity by mimicking the methylated guaninę nucleotide base targeted by MGMT.
6. Concurrent administration of O6BG restores tumor cells sensitivity to TMZ.7. However, O6BG/TMZ administration caused severe off-target myelosuppression (high
incidence of grade 4 neutropenia).
Chemoprotection with gene therapy against chemotherapeutics
1. Expression of the O6BG-resistant MGMT mutant P140K by hematopoietic cellsprovides significant chemoprotection against hematopoietic toxicity from O6BG/alkylator chemotherapy.
2. Prospective phase I/II Clinical trial in which newly diagnosed MGMThi GMB patientswere transplanted with autologous P140K gene-modified HSCCD34+ cells to preventhematopietic toicity duriung combination O6-BG/TMZ chemotherapy.
3. Vector used: gibbon-ape leukemia virus pseudotyped gammaretrowviral vectorencoding the P140K cDNA.
Results: 1. A significant increase in the mean number of tolerated O6BG/TMZ cycles (4.4 cycles
per patient) compared with historical controls without gene therapy (1.7 cycles per patient).
2. One patient tolerated an unprecedented 9 cycles and demonstrated long-term PFS without additional therapy.
3. Biomathematical modeling revealed markedly delayed tumor growth at lowercumulative TMZ doses in study patients compared with patients that receivedstandard TMZ regimens without O6BG.
Chemoprotection with gene therapy against chemotherapeutics (2)
JE Adair, JCI, September 2014
Cancer gene therapy
1. Direct attack on tumor cellsa) transfer of tumor suppressor geneb) suicide genesc) oncolytic viruses (replication-competent viruses)
2. Harnessing immune response to tumor antigens- tumor vaccines- genetically engineered lymphocytes
Immunotherapy of cancer – major facts
Kozlowska et al., Gene 2013
Genetically modified tumor vaccines
Immune system may play a role in controlling tumor growthand development
However, antigens present on tumor cells are not sufficient to boost immune response: - they are weak- lack the co-stimulatory molecules necessary for
full recruitment of cellular immunityMany tumors also employ active immune evasion strategies
hence
Modification of tumor cells – i.e, overexpressing certaingenes, may stimulate immune system to respond to tumor cells
Strategy of genetically modified tumor vaccines
1.Isolate tumor cells from a patient – however, it is oftennot possible to use autologic cells
2. Alternative – culture other tumor cells- eg. cell line of the same type – i.e. allogeneic cell line
3. Transduce such cells with vector – eg. retroviral vectorharboring cytokine gene
4. Inject such modified cells into patients
5. Antigens present on allogeneic tumor cells stimulateimmune system, which respond to the same antigenspresent on patient’s tumor - Cytokines enhance the response
Hyper-interleukin-6 modified cellular vaccine
Kozlowska et al., Gene 2013
Developed against melanoma at the Medical University in Poznan –- prof. Andrzej Mackiewicz et al.
Hyper-interleukin-6 modified cellular vaccine
Kozlowska et al., Gene 2013
H. Ledford, Nature 2014
Immunotherapy of cancer
Chakravarti et al., Trends in Biotechnology, 2015
Adoptive T cell therapy for cancer treatment
Gene therapy for treatment of melanomaCancer Regression in Patients After Transfer of Genetically Engineered Lymphocytes.
Morgan at al.. (Rosenberg), Science, 31 August 2006
Genes encoding TCR specific for a variety of TAA have now been cloned and these include: 1) the MART-1 and gp100 melanoma/melanocyte differentiation antigens2) the NY-ESO-1 cancer-testis antigen present on many epithelial cancers3) the epitope from the p53 molecule, expressed on the surface of approx. 50%
of cancers of common epithelial origin
Tumor associated antigens (TAA) are recognized by T cell receptor (TCR) on the T lymphocyte surface, which is composed of the TCR a and b-chains
Cancer Regression in Patients After Transferof Genetically Engineered Lymphocytes
Peripheral blood lymphocytes (PBL) of the patients with refractory melanoma were transduced ex vivo with retroviral vector encoding T cell receptor (TCR), recognizing:
1) the MART-1 and gp100 melanoma/melanocyte differentiation antigens
Transduction with these TCR encoding retroviral vectors converted normal PBL to cells capable of specifically recognizing and destroying both fresh and cultured cells from multiple common cancers
Those TCR transduced T cells secreted IFN-g following co-culture with melanoma cells
Morgan at al.. (Rosenberg), Science, 31 August 2006
Cancer Regression in Patients After Transferof Genetically Engineered Lymphocytes
Morgan at al.. (Rosenberg), Science, 31 August 2006
Patient 4liver metastasis
Patient 14lymph nodemetastasis
Cancer gene therapy
1. Direct attack on tumor cellsa) transfer of tumor suppressor geneb) suicide genesc) oncolytic viruses (replication-competent viruses)
2. Harnessing immune response to tumor antigens
Oncolytic virus immunotherapy
A therapeutic approach to cancer treatment thatutilizes native or genetically
modified viruses that selectively replicate withintumor cells
Unhindered by interferon-mediated antiviral defence, which is compromised in many tumours, these viruses specifically attack cancer cells by:
1. gaining entry through receptors that are overexpressed in these cells and/or
2. exploiting molecular pathways associated with malignant transformation for their replication
Oncolytic viruses
Replication competent viruses
• autonomously replicating parvoviruses• human reoviruses• vesicular stomatitis virus• Newcastle disease virus
Naturally occuring viruses
Engineered oncotropic viruses
• conditionally replicating adenoviruses (such as ONYX-15)• conditionally replicating herpesviruses (T-VEC)
Oncolytic viruses replicate in cells deficient of p53
Oncolytic virusesLyse only tumor cells, Adenovirus ONYX-015 , with deletion in E1B gene, may amplify
only in cells with mutated p53 gene
E1B 55K protein binds to p53 and blocks its function-wild type virus can amplify in normal cells
In normal cells p53 is active, and lack of E1Bprevents inactivation of p53
Nature
Mechanisms of action of oncolytic viruses
Not fully elucidated, but major ones are:
1. Viral replication within transformed cells2. Induction of primary cell death3. Interaction with tumor cell antiviral elements4. Initiation of innate and adaptive anti-tumor immunity
Mechanisms of viral entry into cancer cells
Kaufman et al., Nature Rev Drug Discovery, 2015
1. Some viruses areable to move via more than one receptor.
2. Some receptors canpromote the entry of more than one type of virus
3. Some viruses useendocytosis to entercells
4. Certain oncolyticviruses are known to preferentially target cancer cells
Improving the potential of oncolytic viruses
3. Oncolytic viruses can also be engineered to take advantage of the abnormal signalling pathways in cancer cells by inserting promoters that are more active in cancer cells or that are tissue-restricted; or regulated under specific conditions, like eg. hypoxia (HRE-elements introduced in the promoters).
4. Arming oncolytic viruses in the tumor-specific antigens: to divert immuneresponse against the tumor, not virus.
5. Combination therapies – oncolytic viruses with immune checkpoint inhibitors.
1. Using tumor-specific promoters: many oncolytic viruses use such proteins whichare aberrantly expressed on tumor cells: eg. HSV-1 uses the herpesvirus entry mediator (HVEM) and selected nectins for cell entry; overexpressed on melanoma and various carcinomas.
2. Engineering oncolytic viruses to target specific receptors on cancer cells; eg. adenovirus Ad5/3-Δ24, which was modified to bind to integrins that are highly expressed on ovarian cancer cells, and is currently being investigated in clinical trials.
Based on Kaufman et al., Nature Rev Drug Discovery, 2015
Ongoing trials with oncolytic viruses
Kaufman et al., Nature Rev Drug Discovery, 2015
53
HSV
Herpes simplex viruses
54
Herpes virus – 1
Advantages
Very large genome – 152 kb – linear, double strand DNA, contains at least 84 genes,of which around half is not required for the virus replication
The virus itself is transmitted by direct contact and replicates in the skin or mucosal membranes before infecting cells of the CNS.
It exhibits both lytic and latent activity. The lytic cycle results in viral replication and cell death. The latent infection allows for the virus to be maintained in the host for an extended period of time.
The virus enters cells by fusion triggered by binding of viral glycoprotein gB and gDto the heparin sulfate moiety of the cell surface protein.DNAenters the cell and circularizes.
Large transgene can be introduced – up to 30 kb
Large number of viral copies can be producedVirus is not toxic, may stay in latent form for a long time
Transduce numerous cell types
Limitations
Lack of data concerning the application of recombinant herpesviruses in patientsProblems with targeting to specific cell type
55
Applications of HSV-1 vectors
Experimental studies
1. Tumors, including nervous systems2. Diseases of the central and peripheral nervous systems3. Injury to spinal cord4. Treatment of pain – delivery to sensory nerves looks particulalry promising
T-VEC – an oncolytic herpesvirus
H. Ledford, Nature 25 Oct 2015
Talimogene laherparepvec (T-VEC, Amgen)
Large clinical trial – melanoma – virus both shrank the tumors in people with advanced melanoma and extendedpatient survival by a median of 4.4 months – R.H. Andtbacka et al., J Clin Oncol 33: 2780-2788, 2015
Recently registered by EMA and FDA
- T-VEC contains deletion of the neurovirulence genes: improves cancer cell selectivity andprevents infections of neurons
- Deletion of another gene (ICP47 – an inhibitor of antygen presentation) results in presentationof viral antigens by both healthy and cancer cells, which leads to the containment of theinfection in healthy tissues and immune mediated destruction of cancer cells that selectivelypropagate oncolytic HSV-1
Anti- cancer gene therapy - summary
1. The largest number of clinical trials in gene therapy arefor cancer
2. Different strategies are employed
3. Experimental and early clinical trials indicate for the potential benefit of this type of therapy
4. Some strategies are already in the phase III clinical trials and show promise to improve the effectiveness of anti-cancertherapy
Exam – planned on 1st February (Monday), room D107, 1 pm