Download Targeting angiogenesis and targeting genomes

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
Hair growth
uterus
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
Somatic
mutation
Small
avascular
tumor
Tumor secretion of
angiogenic factors
stimulates angiogenesis
Rapid tumor growth and
metastasis
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
human VEGF
 RhuMAb VEGF binding is restricted
to human
 Bevacizumab binds VEGF,
preventing interaction with its
receptors and activation of downstream
signalling pathways
 This ultimately leads to vascular
regression, leaving the tumor dormant
X
–P
–P
P–
P–
X
Growth
Proliferation
Migration
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
under the Macula
Wet AMD:Macula with
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
Cardiovascular
diseases
Treatment of
numerous diseases
can be improved by
pro-angiogenic
therapy
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 DNAcleavage 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 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
investigation and forensic medicine.
dramatically
improved
criminal
Recombinant
proteins
Monoclonal
antibodies
Gene
localisation
and function
Gene modification
(mutations)
Transgenic
Animals
Gene therapy
DNA recombination
technology
Molecular
diagnostics
Creation of
new organisms
Forensic
medicine
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