Targeting 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 (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™ () — 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 &

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 other gene therapy methods, which add a functional, or partially functional, copy of a gene to a patient’s cells but retain the original dysfunctional copy of the gene, this system can remove the defect

Long et al. (Olson) Science, Sept 2014 Examples of cell types and organisms engineerd using CRISPR/Cas-9

Doudna & Charpentier, Science Nov 2014 DNA surgeon

E. Pennisi, Science 23 Aug 2013 Mechanisms of Cas-9 action: a way to its modification

R. Barrangou , Science 344: 16 May 2014 Paper to read !

EMBO Mol Med. 2015 Mar 21;7(4):363-5. doi: 10.15252/emmm.201504847.

Freely available in PubMed - http://www.ncbi.nlm.nih.gov/pubmed/25796552 Editing of human genome – discussion

Don’t edit the human germ line

•Edward Lanphier •Fyodor Urnov •Sarah Ehlen Haecker •Michael Werner •& Joanna Smolenski

Nature 12 March 2015 Editing of human genome Biotechnology - achievements

Biotechnology therapeutics approved by the U.S. Food and Drug Administration (FDA) to date are used to treat many diseases, including leukemia and other cancers, anemia, cystic fibrosis, growth deficiency, rheumatoid arthritis, hemophilia, hepatitis, genital warts, and transplant rejection. Biotechnology has created: * more than 200 new therapies and vaccines, including products to treat cancer, diabetes, AIDS and autoimmune disorders. * more than 400 drug products and vaccines currently in clinical trials targeting more than 200 diseases, including various cancers, Alzheimer’s disease, heart disease, diabetes, multiple sclerosis, AIDS and arthritis. * hundreds of medical diagnostic tests for early detection of diseases, for keeping the blood supply safe, or for detection of pregnancy at home. * DNA fingerprinting, which has dramatically improved criminal investigation and forensic . Gene Recombinant Monoclonal localisation proteins antibodies and function

Gene modification (mutations)

Transgenic Animals DNA recombination technology Creation of new organisms

Forensic Gene therapy Molecular medicine diagnostics Applications of genome engineering

P. Hsu et al., Cell June 5, 2014 Last lecture – 19th of May

http://www.biomed2015.pl

Exam – 23rd of June, 13.30 – room D107