Targeting angiogenesis and targeting genomes Lecture 6 – 12 May 2015 Prof. Jozef Dulak Faculty of Biochemistry, Biophysics and Biotechnology Department of Medical Biotechnology Web: www.biotka.mol.uj.edu.pl/zbm New capillary formation in response to wounding Physiological angiogenesis in adults is restricted placenta uterus Hair growth Wound healing Angiogenesis – the formation of new blood vessels from preexisting capillaries Blood vessel formation Vasculogenesis in embryo Vascular endothelial growth factor-A • VEGF-A (vascular endothelial growth factor, vascular permeability factor, vasculotropin) – homodimeric protein, 34-42 kDa • Produced by many types of cells (e.g. macrophages, vascular smooth muscle cells, fibroblasts, and cancer cells) • Expression is induced in response to hypoxia and proinflammatory cytokines • Receptors (VEGF-R1 and VEGF-R2) are present mostly on endothelial cells, therefore VEGF acts specifically on endothelium. Tumor growth is dependent on angiogenesis Tumor growth is dependent on angiogenesis Judah Folkman Tumor growth is dependent on angiogenesis 1933-2008 The Angiogenic Switch is necessary... for Tumor Growth and Metastasis Tumor is dormant Angiogenic switch Neovascularization: Allows rapid tumor growth by providing oxygen, nutrients, and waste removal Facilitates metastasis Tumor secretion of Somatic Small angiogenic factors Rapid tumor growth and mutation avascular stimulates angiogenesis metastasis tumor Carmeliet and Jain. Nature. 2000; 407:249; Bergers and Benjamin. Nat Rev Cancer. 2003; 3:401. Blood vessels in tumors are different than in healthy tissue Figure 13.34b The Biology of Cancer (© Garland Science 2007) Angiogenesis is dependent on the balance between pro- and anti-angiogenic mediators Various strategies to inhibit VEGF signaling Ferrara and Kerbel, Nature 2005 Anti-VEGF antibody Bevacizumab (Avastin) VEGF Recombinant humanised Bevacizumb monoclonal anti-VEGF antibody 93% human, 7% murine Recognises all major isoforms of X human VEGF RhuMAb VEGF binding is restricted to human P– – P P– – P Bevacizumab binds VEGF, preventing interaction with its X receptors and activation of downstream Growth signalling pathways Proliferation This ultimately leads to vascular Migration regression, leaving the tumor dormant Survival Angiogenesis inhibitors in clinical trials …..despite its price, which can reach $100,000 a year, Avastin has become one of the most popular cancer drugs in the world, with sales last year of about $3.5 billion, $2.3 billion of that in the United States. Increase in survival of patients with renal cell cancer treated with Avastin Fever, hypertension, proteinuria – adverse effects Avastin in clinics The FDA approved Avastin in February 2004 for use in combination with intravenous 5-Fluorouracil (5-FU)-based chemotherapy as a treatment for patients with first-line metastatic cancer of the colon or rectum cancer nowadays it is approved for: Metastatic colorectal cancer for first- or second-line treatment in combination with intravenous 5-fluorouracil–based chemotherapy. Advanced nonsquamous non–small cell lung cancer in combination with carboplatin and paclitaxel Platinum-resistant ovarian cancer – in combinationwith paclitaxel, doroubicin or topotecan Advanced cervical cancer – in combination with paclitaxel and cisplatin or paclitaxel and topotecan Metastatic kidney cancer (with interferon alfa) Glioblastoma when taken alone in adult patients whose cancer has progressed after prior treatment. What is the mechanisms of actions of anti-angiogenic drugs? Normalisation of blood vessels as the mechanisms of action of anti-angiogenic agents normal tumor anti-angiogenic therapy Normalisation rather than inhibition of blood vessel formation Jain, Science 2004 Age-related macular degeneration (AMD) Age-related macular degeneration (AMD) Age-related macular degeneration (AMD) is defined as the loss of macular function from the degenerative changes of aging Normal Macula Dry AMD:Drusen formation Wet AMD:Macula with under the Macula abnormal blood vessels The macula is the most important part of the retina responsible for sharp, central vision Current Treatments for AMD… • Lucentis™ (ranibizumab) — The FDA approved Lucentis in June 2006 for the treatment of wet AMD. – Lucentis (ranibizumab) is a humanized anti-VEGF antibody fragment that inhibits VEGF activity by competitively binding with VEGF. – Pegaptanib – Macugen The aptamer-based therapeutic, Macugen, is derived from a modified 2′fluoro pyrimidine RNA inhibitor to VEGF and is now being used to treat the wet form of age-related macular degeneration. Macugen was demonstrated to be effective in prevention of vision loss in two large clinical trials in patients with AMD Tyrosine kinase inhibitors Marty et al. 2008 Angiogenesis may be disturbed in many diseases Treatment of numerous diseases can be improved by anti-angiogenic therapy Treatment of numerous diseases can be improved by pro-angiogenic Cardiovascular therapy diseases www.angio.org Genome editing Scientific American – 12/2014 Świat Nauki – styczeń 2015 Wiedza i Życie – maj 2015 Components of genome editing 1. Targeting tools – recognize specific sequences in the genome 2. Nucleases - cut targeted DNA 3. DNA repair mechanisms – to intriduce changes in the required site Major strategies of genome engineering Programmable nucleases: 1. zinc-finger nucleases (ZFNs) 2. Transcription activator-like effector nucleases (TALENs) 3. RNA-guided engineered nucleases (RGENs) – based on clustered regularly interspaced short palindromic repeats (CRISPR) –Cas (CRISPS-associated)-9 system (CRISPR/Cas-9) Zinc finger nuclease technology Zinc finger nuclease (ZFN) technology utilizes a FokI nuclease as the DNA- cleavage domain and binds DNA by engineered Cys2His2 zinc fingers. Specific zinc fingers recognize different nucleotide triplets and dimerize the FokI nuclease. The activated nuclease introduces a double stranded break between the two distinct zinc finger binding sites, which prompts recombination and modification of the genome http://www.addgene.org/ DNA double strand breaks (DSB) repair mechanisms Hsu et al. Cell, June 2014 Nuclease-mediated double strand breaks reapair system Kim et al. (Lemischka) _ Stem Cell Dev, Nov 2014 TALENs Technology Transcription activator-like effector nuclease (TALEN) systems are a fusion of TALEs derived from the Xanthomonas spp. to a restriction endonuclease FokI. By modifying the amino acid repeats in the TALEs, users can customize TALEN systems to specifically bind target DNA and induce cleavage by the nuclease between the two distinct TAL array binding sites. http://www.addgene.org/ http://taleffectors.genome-engineering.org/ Principle of TALENs design and action (1) RVD – repeat variable diresidue Kim et al. (Lemischka) _ Stem Cell Deve, Sept 2014 Principle of TALENs design and action (2) http://www.creative-animodel.com/Animal-Model-Development/ Applications of TALENs http://www.creative-animodel.com/Animal-Model-Development/ E. Pennisi, Science 23 Aug 2013 CRISPR – clustered regularly interspaced short palindromic repeats Natural mechanism of microbial CRISPR system in adaptive immunity Hsu et al., Cell, June 2014 Biology of the type II-A CRISPR-Cas system crRNA – CRISPR RNA Doudna & Carpentier &, Science Nov 2014 Towards the RGENs based on CRISPR/Cas-9 Doudna & Carpentier &, Science Nov 2014 Towards the RGENs based on CRISPR/Cas-9 http://www.hhmi.org/news/jennifer-doudna-shares-breakthrough-prize-life-sciences CRISPR Technology RNA-guided endonucleases (RGEN) utilize a short guide RNA (gRNA) to recognize DNA, bind an endonuclease, and induce site specific cleavage. New RGEN technologies are popularly referred to as CRISPR systems, derived from the clustered regularly interspaced short palindromic repeats (CRISPR) found in bacteria that serve to identify and destroy foreign DNA. CRISPR genome editing systems allow users to design gRNA which target their DNA sequence of interest. When expressed intracellularly in conjunction with a CRISPR associated endonuclease (Cas9), the gRNA directs Cas9 to the target sequence where it unwinds and cleaves the double stranded DNA. The CRISPR genome editing systems are comprised of only 2 to 3 plasmids, expressing the gRNA and the Cas9 nuclease. These systems are easily tuned for targeting specificity by inserting a complementary oligo into the gRNA expression vector. Additionally, various CRISPR systems for genome editing have been developed for use in different cell types. Addgene.org Mechanisms of CRISPR/Cas-9 action Addgene.org CRISPR/Cas-9 editing by non-homologous end joining repair (NHEJ) Addgene.org CRISPR/Cas-9 editing by homology directed repair (HDR) Addgene.org Development and potential applications of CRISPR-cas-9 system Emmanuelle Charpentier & Jennifer Doudna The Breakthrough Prizes recognize pioneering work in physics and genetics, cosmology, and neurology and mathematics. Each prize carries an award of $3 million Doudna & Carpentier, Science Nov 2014 Future applications of CRISPR-Cas-9 Doudna & Carpentier &, Science Nov 2014 Comparison of ZFNs and TALENs K. Gammon, Nature 13 November 2014 CRISPR-Cas-9 K. Gammon, Nature 13 November 2014 Comparison of three classes of programmable nucleases /CRISPR/Cas-9 H. Kim & JS Kim, Nature Rev Genetics , May 2014 Genome engineering using CRISPR-Cas-9 Nature Reviews Microbiology Genome editing and gene therapy of human diseases Repair of mutation in diseases such as: - Duchenne mucular dystrophy - sickle cell anemia - other monogenic diseases Unlike