Leaders in Pharmaceutical Business Intelligence (LPBI) Group

LIVE 9/19 1:40 – 3:20 EMERGING APPLICATIONS OF CRISPR/CAS9 at CHI’s 2^nd Annual Symposium CRISPR: Mechanisms and Applications @ CHI’s 14th Discovery On Target, 9/19 – 9/22/2016, Westin Boston Waterfront, Boston

 

CHI’s 2^nd Annual Symposium CRISPR: Mechanisms and Applications @ CHI’s 14th 

Discovery On Target, 9/19 – 9/22/2016, Westin Boston Waterfront, Boston

http://www.discoveryontarget.com/

http://www.discoveryontarget.com/crispr-therapies/

#BostonDOT16

@BostonDOT

 

Leaders in Pharmaceutical Business Intelligence (LPBI) Group is a

Media Partner of CHI for CHI’s 14^th Annual Discovery on Target taking place September 19 – 22, 2016 in Boston.

In Attendance, streaming LIVE using Social Media

Aviva Lev-Ari, PhD, RN

Editor-in-Chief

http://pharmaceuticalintelligence.com

COMMENTS BY Stephen J Williams, PhD

1:50 MicroRNA Target Site Editing of Chondrocyte Master-Regulators in Primary Human Cells Using CRISPR-Cas9

Christine Seidl, Ph.D., Post-Doctoral Research Associate, Cell Signaling, Kennedy Institute of Rheumatology, Oxford University

 

Osteoarthritis -most common for in UK

33% of people 45 years older

49% of women over 75 years

In US 1 in 2 will have knee OA

Chondrocyte: hyalronic acid, chondroitin

The problem in OA is more breakdown of these proteins than synthesis

Some of these governed by microRNA

Microrna changes during cartiledge differentiation

They found noncoding RNA during cartiledge degradation important

microRNA and RNA silencing complex

Mir675 cola2 mmp13

mir 145      sox9

Mir 138       Hif2alpha

Mir140      adamts5

 

• Need selectivity: avoid manipulation miRNA but mutate the miRNA target at DNA level
• Chondrocyte experiments are tricky since the primary cell type quickly differentiates into a fibroblastic phenotype
• Use PCR barcodes to determine if targeting is specific
• So you use a ribonucleotide protein (RNP with cas9 to make the cas9 guide RNP complex
• Transfection ofguide RNA
• RNP transfection delivers to the DNA
• They wanted to target sox9 which is upstream of aggrecan and col2a1
• Cartiledge is hypoxic so have to work in hypoxic conditions; mir145 downregulates sox9
• Use CRSPR to uncover functional and individual microRNAs

 

#inflammation

@UniofOxford

#CMT

#geneediting

#CRISPR

#Cas9

#NIH

#CMT

#drugdiscovery

#pharmanews

#CRSPR

#genomics

 

@BiotechNews

@PharmaNews

@pharma_BI

@AVIVA_1950

@DrugDiscover365

2:20 Massively Parallel Combinatorial Genetic Perturbation Screening with CRISPR-Cas9 in Human studies

Cheryl H. Cui, Ph.D. Candidate, Harvard-MIT Division of Health Science and Technology, MIT

 

With annotationof the genome is critical for developing the field of genome screening

• With barcoded based assays can increase the library size dramatically
• Limitation of combinatorial studies: hypothesis driven, plasmid co-transfection tricky, pooled PCR stitching method
• Limits the combinatorial high throughput
• COMBIGEM from MIT: pooled digestion plasmid library
• Pooled digest of barcoded clones and can reconstruct them in a lentiviral based library for infection
• They made a barcoded gRNA library to use for screening
• They reported knock out of two genes at same time (8 day post infection); get a multiplexed CRSPR
• Dual gene repression occurred in a single cell
• Useful for combinatorial drug ? NCI estimates 4 million $ to fully screen their nci60 pael
• Used cell proliferation as their screen after transfecting OVCAR8 with their COMBIGEM library
• Question: how do you know that you are not targeting an epigenetic mechanism; found targets and validated with two known drugs so look for targets and validate with drugs, ? but drug usually have multiple targets?
• Could increase bioproduction of vaccines? possible

 

We are going from single-gene to paired gene studies

 

TALK SPECIFIC # and @

 

#systems_biology

#genetics

@MIT

@Harvard

#genomics

2:50 The Scientist’s Guide to CRISPR Law

Paul Enríquez, J.D., LL.M., Ph.D. Candidate, Structural and Molecular Biochemistry, North Carolina State University

 

Case 2013: Myriad Genetics – can genes be patented; Justice Scalia “joined the court but”… did not believe the court’s judgement even though did not know anything about the molecular biology

 

Anthony Kennedy – “ I thought cDNA was an kind of an economy class gene”

 

James Watson:  homosexualtiy, stupid people etc

 

Kary Mullis – invented PCR but “little evidence HIV causes AIDS”

 

Law is not directed by impericism – there is no CRSPR law yet!

 

USDA, EPA and FDA have authority over DNA derived GMO for example so possible anything a CRSPR scientist does could be under this jurisdiction

 

Researchers at Penn State trying to grow CRSPR crops and USDA came in and said no reason to believe CRSPR crop is not a pest… first time CRSPR got a regulatory pass

 

NIH trying to release bacteria in 1985 genetically recombined and second circuit court came in and stopped it

Oxytec was making a transgenic mosquitos in Brazil Panama with gene-driven inheritance (altered gene preferentially inherited) HOWEVER senate democrats voiced concern when they tried to release them in Florida

 

CRSPR animal research guidelines   

Genome wide inactivation of PERV (George Church): United Technologies signs $100 million to develop humanized pigs

 

WOW every state in US has stem cell clinic BUT NOT REGULATED by FDA!!!

 

In 2014 FDA defined stem cells as a drug –  title 21 so CRSPR could fall under this title

 

George Church initially was against germ line editing but now has changed his mind

NIH greenlights a CRSPR trial in human cancer if FDA gives them the green light

Constitutional law sometimes follows the mob e.g. sterilization of mentally challenged in 1928

#FDA

#CRSPR

#regulatory

@NCarolinaSt

EMERGING APPLICATIONS OF CRISPR/CAS9

1:40 Chairperson’s Opening Remarks

James Inglese, Ph.D., Head Assay Development & Screening Technologies, National Center for Advancing Translational Sciences, National Institutes of Health

1:50 MicroRNA Target Site Editing of Chondrocyte Master-Regulators in Primary Human Cells Using CRISPR-Cas9

Christine Seidl, Ph.D., Post-Doctoral Research Associate, Cell Signaling, Kennedy Institute of Rheumatology, Oxford University

MicroRNAs (miR) are important regulators of gene expression. Frequently, several potential target sites are located on a transcript but only one constitutes the dominant regulatory element. In this talk, a method will be discussed that allows for endogenous miR target site identification in primary human chondrocytes using CRISPR-Cas9 without the need of clonal selection of edited cells.

• chondrocyte only cell type with articular cartilage – change in gene expression pattern:

1. Protein coding genes
2. non-coding RNA

• MicroRNA generation and theur function: Seed sequence target site
• Colagene – COL2A1, MMP-13 (mouse)

1. Transcription factor – need selectivity – effect mRNA
2. CRISPR-Cas9 : Cartilage Chondrocyte with cartilage matrix — enzymatic transfer
3. Plasmid transfection not possible
4. In vivo by site-specific genome engineering
5. Barcode tagging by T7 / T3 primer sequence insertion – bDNA vs RNA: Silent MTS vs active MTS
6. RiboNucleoProtein (RNP) –>> Cas9 >> single guide RNA (cr/tracr) –.. Cas9-gRNA by RNR >90% viability of HACs
7. TF Cas9-RNR – Cas9 – RNAPII
8. Hypoxic env: Cartilage – avascualar, low oxygen tension: MiR-145 regulate SOX9

2:20 Massively Parallel Combinatorial Genetic Perturbation Screening with CRISPR-Cas9 in Human Cells

Cheryl H. Cui, Ph.D. Candidate, Harvard-MIT Division of Health Science and Technology, MIT

The systematic analysis of combinatorial gene functions is labor-intensive and challenging to scale. Our platform enables massively parallel screening of barcoded combinatorial gene perturbations in human cells. This technology leverages the simplicity of the CRISPR-Cas9 system for multiplexed targeting of specific genomic loci and the scalability of CombiGEM (Combinatorial Genetics En Masse)-based DNA assembly to construct barcoded combinatorial genetic libraries that can be quantified with high-throughput sequencing.

• Annotation and Screening Libraries
• Barcoded-based pooled screening
• high throuput NGS

1. Engineering cell behavior – gene by gene aproach, gene combination
2. plasmid co-transfection or multiple viral infection
3. pooled Combinatorial Genetics En Masse (CombiGEM)

• Barcoded of assorted genetic elements
• Barcoded (n)-wise combinatorial library
• Programmable Genetic Pertubations
•  Oligonucleotide array: Lentiviral vector: Guide sequence A and B

1. Deep sequencing of targeted genomic loci
2. immunoblot analysis of targeted protein targeted genomic loci sequencing in single cell

• DIscovery and Drug COmbination: NCI tested 5000 Pair-wise
• Epigenetic states are reversible
• Global alteratuons of epigenetics landscape
• gRNA Libraries targeting epigenetics regulators –
• Lentiviral delivery of barcoded combinatorial tat inhibit cancer cell growth of Ovarian Cancer cells
• CombiGEM-CRISPER results are reproducible
• synergistic suppression of ovarian cancer cells with drug combination

Summary

• massive parallel genotype-to-phynotype ampping in human cells
• high reprodicibility Broad applications of CombiGEM in Human cells
• Mapping and Deciphering dynamic and Combinatorial Genetic Networks
• use combinatorial genetic networks for drug discovery

2:50 The Scientist’s Guide to CRISPR Law

Paul Enríquez, J.D., LL.M., Ph.D. Candidate, Structural and Molecular Biochemistry, North Carolina State University

CRISPR systems are revolutionizing science and biotechnology. However, great uncertainty exists surrounding how the law will treat this nascent biotechnology. This talk provides an overview of the key regulatory issues every CRISPR scientist should know. Drawing parallels from stem cells and gene therapy, the talk highlights the importance of law and policy in fostering CRIPSR-based research, and makes recommendations for scientists studying CRISPR mechanisms and applications.

• GMO – can cause Cancer
• Hacking Genomes: Gene driven inheritance – Trandgenial musquitos – Oxitec – bite human
• Endogenous modifications – crop advantage
• Endogenous modifications – confer pest resistance
• CRISPR in animal research – NIH – ban use of Monkeys for researrch funded by NIH – Animal Welfare right
• autism-like behavior replicated in Porcine
• development of human organs in pigs
• Stem cell tourism – Stem cell Clinic not regulated by FDA
• Gene therapy by CRISPR
• FDA Jurisdiction: COsmetic Act, Public Health
• Human Germ Line gene modification

 

3:20 Close of Symposium