LIVE 9/20 8AM to noon GENE THERAPIES BREAKTHROUGHS at 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 14th Annual Discovery on Target taking place September 19 – 22, 2016 in Boston.
In Attendance, streaming LIVE using Social Media
Editor-in-Chief
http://pharmaceuticalintelligence.com
COMMENTS BY Stephen J Williams, PhD
Gene Therapy Breakthroughs
New Strategies for Better Specificity and Delivery
2:05 Chairman’s Remarks
Joseph Gold, Ph.D. Director Manufacturing Center for Biomedicine and Genetics, Beckman Research Institute City of Hope
- CBG (center for biomedicine and Genetics) 20000 sq feet
- CTPC (center therapy production) mainly CART
- CBG 16 years operation do all stem cells and >400 products
- New stem cell Beta cell progenitor
- Do oncolytic VSV
- CTPC is investigator driven CART islet cells,
- Like to do novel work so work with CIRM
- Banking of modified stem cells
- Adherent scale out limitations: cost,inefficient; solution can be suspension
- Establish hESC; plate on CELLstart > Accutase>StemPRO SFM>differentiation process; defined reagents — they use this for cardiomyocyte differentiation: they are functional (inotropy, chronotropy response to isoproterenol) can freeze back cells
- Create a bank of intermediate cells and when you need it for surgery they will put on their matrix, enrich, expand and ship out
- Allogeneic cells: project where take allogeneic neural stem cells to deliver a chemotherapy payload as they like to migrate to brain tumors
- Allogeneic cells: for ALS modified to express GDNF
- HIV resistance with engineered CCR5 negative blood stem cells
- Release assay considerations: viability, sterility, if cryopreserved then can determine identity, viral insertions, VSV-G copy number, endotoxin and potency (FDA is wanting phase I potency assays) for CART potency is % transduced
- Good in vivo activity of the neural stem cells loaded with chemotherapeutic
ALS
- If deliver GDNF to muscle using genetically modified myoblasts
- Best to use fetal stem cells – less issues
Canavan disease: progressive fatal neurologic disorder that begins in infancy and don’t make it past teenage years
- Rossbach is taking autologous cells reprogramming generating iPS cells and then modifying by CRISPR but the CRISPR issues of off target effects persist as well the time required for process and verification; also don’t want to use a selectable marker and put in patients; so you can differentiate the cells and hit them with a lentiviral vector system
They have been named a PACT Center Production Assistance for Cell Therapy where you can apply for a project grant. Applicable for startups up to larger mature companies
www.pactgroup.net
They do a standard panel of tests for viral infections.
They work with investigators or companies at all stages of manufacturing processes.
@BeckmanInst
@cityofhope
2:15 Large-Scale Production of Cell Therapies for Regenerative Medicine
Joseph Gold, Ph.D. Director Manufacturing Center for Biomedicine and Genetics, Beckman Research Institute
2:45 Directed Evolution of New Viruses for Therapeutic Gene Delivery
David Schaffer, Ph.D. Professor of Chemical and Biomolecular Engineering, BioEngineering, Molecular and Cell Biology and Neuroscience;
AAV is very safe as many people already infected with it
- Spark (Leber’s cogenital anaurosis
- Hemophilia B
- Lipoprotein lipase deficiency
- Spinal muscular atrophy
- Challenges: are we just getting the ‘low hanging fruit’ eg Spark therapy must be injected after retinal therapy, hemo B needs to be given at high doses
- Their theory was AAV had been evolving for its own purposes so hence the limitations of AAV;
- Utilized 25 different techniques to generate variants of AAV in a library then packaged (each will have its own barcode)
- Broad platform technology: retina, lung, brain and spinal cord
Retinal: AAV may be too large to get through layers of the eye, problems; subretinal injections and damage or retinal detachment. Then they used their whole library in an in-vivo screen (as hard to recapitulate the multi cell layers of the retina).
Cystic Fibrosis
- AAV2H22 variant worked very well to supply the CFTR gene in pig model of cystic fibrosis and increases chloride transport and reduce bacterial load
- Then found pig variant AAV did not work well on human tissue so designed a human variant and worked well in human tissues
- The variant AAV2.5T surrounds sialic acid binding pockets and increases binding and endocytosis
Brain and Spinal Cord: Sanfilippo B trial 8 holes drilled into skull followed by 16 AAV injections
- Injected a generated AAV variant (by evolution process) : engineered AAV2 is 100 fold better getting through blood brain barrier… novel variant undergoes retrograde transport to cortex ; made a cas9 to remove a tdTomato gene overexpressed in mouse and found 90% knockdown
- Also interesting point: the porcine variant did not work in human and the human variant did not work in porcine. Implication for FDA safety and efficacy testing must do in monkeys
They started a spinout 4D Molecular Therapeutics
4:25 Lentiviral Vectors for Gene Therapy
Munapaty Swani, Ph.D. Texas Tech Health Science Center
- Can express multiple shRNA under a separate promoter but toxic so if expressed in miRNA backbone could be safer under a pol II
- How much of flanking sequence is needed?
- 30 nt flanking sequence is enough for Drosha processing
- Constructed 1 to 7 shRNA-miR targeting CCR5 and 6 viral genes; all constructs were functional
- Problem with pol ii promoter
- These 7 shRNA miRNA protect against HIV entry if against CCR5 and the 7 viral elements
- Used the non-integrating lentivirus for transient to see if infect T cells or not versus integrating lentivirus ; results non-integrating lentivirus did not infect t cells so safer to use
- CCR5 disruption reduced HIV infection in T cells in vitro;
- ZFN treatment of HIV+ PBMC prevents activation of HIV
- Encapsulted CAS9 within LV; cas9 protein is incorporated within LV and is functional
- First transduce then come in with the Cas9 so made all in one lentivirus with Cas9 and an sgRNA expression vector *******
- This shows that it is possible to put all in a nanoparticle based lentivirus and an all in one may make it easier and safer (supposedly)
4:55 AAV Capsid Design
Miguel Seria Esteves, PhD Associate Professor Neurology, Gene Therapy Center University of Massachusetts Medical School
-AAV replication dependent no known human disease with native AAV
- Multiple barriers to get across blood brain barrier
- AAV9 preferentially target neonatal neurons and adult astrocytes
- Multiple capsids can be used for AAV9 infection in brain but not complete
- Can we design better capsids to give it better tropic properties and better penetration to blood brain barrier
- Using a polyalanine in the 5’ end of the caspid was most efficeint
- Increases gene transfer efficiency especially IN SELECT CELL TYPES; Glial transduction and increased in striatum: increase is structure specific so little in thalamus but good in cerebellum and spinal cord
- AAV9 tranduces also in peripheral tissues with or without modified capsid
Huntington’s Disease
- Polyglutamate disease polyy glu on huntingtin protein
- They get a 40 to 50% reduction of huntingtin but not significant between capsid design
- They did a directed evolution of AAV capsid and generated capsid gene delivery diversity: DNA shuffling and in vivo selection
- AAV-B1 is a new tropic capsid showing transduction of different structures
- Five fold reduction in tropism to the liver but massive increases in muscle and beta exocrine cells and lung
- Presence of neutralizing antibodies is a problem with AAV therapy
- In conclusion unknown mechanisms by whivh a highly hydrophobic string of 19 alanines modifies the CHS tropism of AAV9 kvariants
- Chimeric capsids identified from in vivo screen can reveal interesting patterns of tropism
8:20 AAV for Gene Therapy and Genome Editing
James Wilson, M.D., Ph.D., Professor, Department of Pathology and Laboratory Medicine, Perelman School of Medicine; Director, Orphan Disease Center and Director, Gene Therapy Program, University of Pennsylvania
AAV delivery for CNS can be direct into brain or into CSF but AAV are big and vectors usually don’t cross BBB
Mucopolysaccharidoses a lysosomal storage disease including Hunter, Morquio
- There are canine models for these diseases
- Data show that intrathecal delivery has good distribution to the CSF not the serum
- With IV you only get spinal distribution but IT you get good distribution to cerebellum and frontal cortex
- Intrathecal superior for clearing lesions in cortex of dogs
- In the dog the disease is more skeletal and less neurologic so with IT dogs were better than control but still some problems
- Avaxis: AAV9 gene therapy for SMA and ALS but trials are very small but seems to be dose dependent effect ; phase I/II done; Pfizer, Esteves and REGENEXBIO have trials
- Liver transduction of AAV has always been a success; early vectors were not efficient for uptake but AAV9 hemophelia B (factor 9) was
- OCTD disease of urea acid cycle (neonatal versus late onset); proteins altered metabolism
- Have to create a metabolic sink to detoxify metabolites; not lie a gene replacement therapy
- They got stable expression in mouse adult model of OCTD with AAV and had rapid onset of expression; but when done in newborn mice they saw transient expression
- Newborn liver is proliferating so gene vector may be diluted out versus the adult liver
- So turned to gene editing (ZFN:NEHJ, TALEN:HR gene correction, CRSPR:transgene addition by HR
- Staph aureus cas9 is smaller than most and can fit in a 4.79kb vector*****
- Put in 2.6 kb doner OCTD gRNA
- With a CRSPR-Cas9 mediated deliverycould maintain the expression of OCTD for over a week in newborn mice
- BUT in adults the gene corrected animals started to die; they were losing their ability to break down protein
- In newborns you got 10% gene editing with 30% indels but with adults 30% resulted only in 1% gene editing
- There is a propensity to create large (>50bp) indels in the adult
- NGS was needed to fully detect the target transgene integration, PCR is not good enough
- Says that large animal models are needed for safety/efficacy studies
- Problem with Rhesus monkey: started with a humanized mouse in Rag mice because did not want to do monkey studies; but did not get good expression in the monkeys (it was not the vector which was the problem)
- AAV may be good enough of a donor to cause the HR recombination
Summary: AAV vectors combined with a CRSPR CAS9 system is effective in neonatal delivery however 1) AAV by itself may be a good delivery system by itself but need the crsipir guide RNA to make the break to promote HR and get the best efficiency of integration 2) use of CRSPR CAS9 may direct the proper integration you want to deliver the OCTD exons to correct gene loci
Talk specific @ and #
#genetherapy
#virology
@PennMedicine
9:20 Using CRISPR/Cas9 to Target and Destroy Viral DNA Genomes; Inactivating HBV
Bryan R. Cullen, Ph.D., James B. Duke Professor of Molecular Genetics and Microbiology and Director, Center for Virology, Duke University
The CRISPR array is an evolutionary record of the bacteriophage that the bacteria have encountered.
- Incredible that a bacteria that never encountered a chromosomal structure would scan the genome with these gRNAs and then initiate HR and NEHJ (error prone in mammalian cells usually 3 nt
- HBV is a very confused retrovirus because it first goes into the nucleus then becomes a template for RNA synthesis to make the particles that reinfect the cell and invisible to immune response
- Although they do not produce infectious virus it still remains in genome
- sgRNA screening: use a luciferase based assay to correct or knock out luc so looking for decrease of luciferase activity
- In a model of HBV infection where they have an inducible HBV cccDNA in a single integrated copy the cas9 reduced the DNA but protein was not decreased that much (if you hit episomal DNA you see loss and the disintegration of cut out DNA but if you hit integrated DNA you see repair
- In their case the integrated DNA was just mutated (A or AA insertion) so in essence a frameshift mutation
- Future strategies: use two guide RNAs to permit deletions or allow use of nickase. Currently these gRNA use polIII which is very large
- They are editing the VEGF loci using Sau Cas9 and two sgRNAs
- In mice with HBV integrated in their genome get some cutting but they need to go to higher doses of Cas9 system
- Potential future success if reduce viral load as HBV continually release antigen which results in T cell anergy and if reduce the viral load may help to reduce anergy and wake up the immune system
Meeting specific # and @
| #genetherapy
#virology
#adenovirus |
@Duke |
10:35 Targeted Endonucleases as Antiviral Agents: Promises and Pitfalls
Keith R. Jerome, M.D., Ph.D., Member, Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center; Professor and Head, Virology Division, Department of Laboratory Medicine, University of Washington
- Can cure Hepatititis C because can stop replication
- HSV in neurons and live for life: long lived form like HBV
- Herpes simplex (HSV) establishes in dorsal root ganglion: acyclovir might be useful but came off patent
- He reached out to advocacy groups to get data on need for HSV cure to convince funding agencies this is important
- They found it is important to people; 90% want a cure if 5 years away
- Homing endonucleases: small 800bp high specificity easy to put in vectors more difficult retargeting to other DNA targets
- www.ltk.uzh.ch/de/dyn
- HSV homing endonuclease from Cellectis AG targets a 24bp sequence in Ul19; introduces a DSB at target site with 4 bp 3’ overhang;
- AAV targeted endonuclease delivery; nonimmunogenic; persists in episomal state
- Exposure to HSV specific HE decreases virus production from neuronal cultures at all stages of replication cycle; if infect and let go for a month but the HE disrupts HSV in acute late-acute and late cycle
- Developed a mouse model of HSV infection and AAV delivery in vivo system; so the AAV was accessing the nerve endings and going down to the trigenital dorsal ganglia; transport is independent of AAV serotype but transgene expression is HIGHLY dependent on AAV serotype
- The in vivo HE treatment appears well tolerated however just a casual observation
- Used NGS with bioinformatic approach to determine off target sites for the most likely mouse loci
- HE suppresses viral reactivation (used PCR based reactivation assay)
Talk Specific @ and #
#drugdelivery
#genedelivery
#AIDS
#genetherapy
#HIV
@fredhutch
@defeatHIV
12:05 pm CRISPR/Cas9 for the Screening of the Human Kinome – A Pilot Study in an Aggressive Pediatric Cancer Cell Line
Simone T. Sredni, M.D., Ph.D., Research Assistant Professor, Neurological Surgery, Northwestern University Feinberg School of Medicine, Ann and Robert H. Lurie Children’s Hospital of Chicago
LentiArray CRISPR Kinase Array
- Malignant rhabdoid tumors (MRT); among most aggressive of pediatric tumors rare but lethal
- Inactivating mutations in SMARCB1 (INI1 gene)
- Component of swi/snf chromatin remodeling complex
- Can originate anywhere in kidney brain and spine
- Kinase inhibitors may be effective (PLK1, ERBB2, AURAKA) : AURKA inhibitor in phase 2 and giving promising response
- Used LentiArray viral vector system and stable expressed Cas9
- Tested 160 kinases in screen; high transfection efficiency
- PIMs; protooncogenes (proviral common integrations sites in Maloney leukemia; overexpressed in prostate and hematologic; effects cell cycle cdc25a is a target and cell survival targets as well; PIM3 high copy number in MRT as well as PIM2
- KO limits proliferation increases senescence and confirmed by MTT with pan PIM inhibitor CS6258 (Cyclene Pharmaceuticals)
- PLK4 overexpression in peds haploinsufficient mice do make tumors; is it mutated? Yes inactivating mutations
- PLK4 expression higher through cell cycle – dependent?
- KI67 was down with Knockdown
- Decrease in clonogenic assay on plastic not soft agar
- CFI-400945 is PLK4 inhibitor is ovarian trials
- Is effective in MRT and comparable to their cas9 knockdown
ssreni@luriechildrens.org
@NorthwesternMed
#cancer
#kinome
#brain
#ChildhoodCancerAwareness
GENE THERAPIES BREAKTHROUGHS
Tuesday, September 20
7:00 am Registration Open and Morning Coffee
8:05 Chairperson’s Opening Remarks
Bryan R. Cullen, Ph.D., James B. Duke Professor of Molecular Genetics and Microbiology and Director, Center for Virology, Duke University
8:20 AAV for Gene Therapy and Genome Editing
James Wilson, M.D., Ph.D., Professor, Department of Pathology and Laboratory Medicine, Perelman School of Medicine; Director, Orphan Disease Center and Director, Gene Therapy Program, University of Pennsylvania
In vivo delivery of nucleic acid therapeutics remains the primary barrier to success. My lab has focused on the use of vectors based on adeno-associated virus (AAV) for achieving success in pre-clinical and clinical applications of gene replacement therapy. Most of the current academic and commercial applications of in vivo gene replacement therapy are based on endogenous AAVs we discovered as latent viral genomes in primates. These vectors are reasonably safe and efficient for application of gene replacement therapy. The emergence of genome editing methods has suggested more precise and effective methods to treat inherited diseases in which genes are silenced or mutations are corrected. AAV vectors have been the most efficient platform for achieving genome editing in vivo. We will review our attempts to achieve therapeutic genome editing in animal models of liver disease using AAV.
- AAV for delivery of vector to CNS
- Novel AAV Platform – capsid platform – distributed by PENN Vector
- direct
- IV
- into CNS: Head and Spinal MPS – Mucopolysaccharidosis – Class of lyposomal Storage Disorder
- AAV9: Binds Glycans with Terminal Gal – BBB
CNS gene transfer in canine model of MPS VII following IV and intrathecal AA( administration
- Serum
- CSF: Frontal cortex, Cerebelum, Spinal Cord
- Intravenous (IV) and Intracisternal (IC) – AA9
Gene Therapy for Motor Neuron Disease: SMA and ALS: PhaseI/II clinical trial of AAV9-SMN IV in infants with SMA1 – Nationwide Children’s
- High dose +23.3 Month survival
Summary
- Avexis
- Abeona
- Pfizer
- Nationwide
- Esteves
- REGENXBIO
Liver Transduction Following IV Adm of AAV8 Vectors
- Mouth liver Day 3 vs Day 90
- Hemophilia B: Therapeutic protein
Urea Cycle Disorder
- newborn OTCD infant in HA crisis
Goals: efficient but Transient vorrection following AAV* Gene Therapy in Newborn
transfer of caspid – promoter vector to correct diffect
- Survival: neonatal gene therapy – two doses of vector, three injections – immunogenic
- Liver transplantation of neonatal before 1 year of age
Gene Editing
- gene targeting by ZFNs, TALENs or CRISPR/Cas9
- WT donor DNA
- Transgene donor DNA
- Gene disruptions
In Vivo correction – liver mouse by AAV. CRISPR-SaCas9
- OTC donor template
- efficient restoration of OTC Expression in the liver – mice treated at Neonatal Stage by AAV8.CRISPR-SaCas9 – Vector administration
- Cas9 – Kinetics of Cas9 – Week 1,3,8 – dilutes with time
- Should work in Adult mice vs Neonatal mice: Low dose vs High dose
- Ureogensis increased
- On-target Deep Sequensing and their Distribution in Neonatal-treated and Adult-treated Animals
In vivo gene editing: mixed results: Site-directed Insertion of hOTCco Gene Cassette in the OTC gene : Controls: WT and spf(ash)
Western Blot: NGS analysis demonstrates on target transgene integration 20 to 32%
High Protein DIet to Evaluate the Efficacy
- NEW BORN – Gene Targeting in FIX-KO Mouse by CRISPR/Cas9: Infron1, Exon 2 Infron2 Exon 3
- ADULTS – Gene Targeting in FIX-KO Mouse by CRISPR/Cas9:
Gene therapy must accumulate experience in Animal models: Safety and Efficacy
NEGATIVE RESULTS IN MONKEYS: Analysis of Liver Tissue for Editing and SaCas9
- CRISPR/Cas9 -mediated Gene knock down of rhPCSK9 in Monkeys (Rhesus Macaque) in LIVER
- In vitro sgRNAs in monkeys and human cells
- EGFP sgRNA vs hrPCSK
- In vivo does not infer In vitro
- Gene Editing in Human — we are not yet there
9:20 Using CRISPR/Cas to Target and Destroy Viral DNA Genomes
Bryan R. Cullen, Ph.D., James B. Duke Professor of Molecular Genetics and Microbiology and Director, Center for Virology, Duke University
A number of pathogenic human DNA viruses, including HBV, HIV-1 and HSV1, cause chronic diseases in humans that remain refractory to cure, though these diseases can be controlled by antivirals. In addition the DNA virus HPV causes tumors that depend on the continued expression of viral genes. Here, I will present data demonstrating that several of these viruses can be efficiently cleaved and destroyed using viral vectors that express Cas9 and virus-specific guide RNAs, thus providing a potential novel approach to treatment.
- HBV – as target intervention
- CRISPR/Cas9 RNA Guided Nuclease System (RGN)
- NGG- PAM >> dsDNA cleavage >> NHEJ Repair >> Prfect repair >> Completion Repair Cycle
- vs Mutagenesis – cycle exit
- HBV – Inactivation with CRISPR/Cas9: HBV lifecycle of the virus — reverse transcripatse (RT) of caspid and envelope – release antigens in blood invisible to Immune response, SUrfacce Antigen,COre Antigen, X-protein,
- Inhibitors of HBV – cccDNA is stable for decades – virus in blood do not create new virus
- tetracycline represses HBV expression
- HBsAG va HBeAG: on core, surface, RT and N.S. sgRNA
Vector Delivery Strategies to the Liver
- Strep pyogenes Cas9 – a diffrence PAM (5′ – NNGRRT-3′) – too large – ~4.8kb, including th ITRs.
- Can we identify smaller Promoters
- Packaging Sau Cas9 and two sgRNAs into AAV
Summary
- using HBV-infected hepatocytes of a humanized liver or transgenic mouse – HBV Dual Target
Using HBV with RT and CRISPR Cas9 — viral load reduced to awake the immune response – allergy stage – potential for future therapeutics
9:50 Grand Opening Coffee Break in the Exhibit Hall with Poster Viewing
10:35 Targeted Endonucleases as Antiviral Agents: Promises and Pitfalls
Keith R. Jerome, M.D., Ph.D., Member, Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center; Professor and Head, Virology Division, Department of Laboratory Medicine, University of Washington
Genome editing offers the prospect of cure for infections such as HIV, hepatitis B virus, herpes simplex, and human papillomavirus, by disruption of essential viral nucleic acids or the human genes encoding receptors needed for viral entry. This talk will highlight the most recent laboratory data and the challenges still ahead in bringing this technology to the clinic.
- Anti HSV – episonal DNA in Neuron, Acylovar – willingness to Participate in studies
- Anti-HBV –
- cART – HIV –
Viral replication returns if medication is taken away in two weeks
Herpes Simplex Virus HSV -1 – 50% HSV-2 16%
- CRISPR/Cas – difficult vectorization
- Targeted endonucleases
- Rare-cutting endonucleases in Gene Therapy – target distruction
- Derived from Crel enzyme by CELLECTIS AG (Paris) – co-expressed with Trex2 to remove 3″ hangover
- AAV as a targeted endonuclease delivery vector – mediated delivery to primary neuronal cultures
- Exposure to HSV-specific HE decreases virus production from neuronal cultures – virus production in treated cells
- HSV-specific HE can disrupt HSV at all stages of the replication cycle – established in vivo
- cell planting
- AAV-mediated transgene delivery to the mouse TG in vivo – injection whiskerpad – eye scarified vs Eye not scarified
- AAV serotype: Transgene expression in TG is highly dependent on AAV serotype
- Dose dependence – Trigeminal ganglion (TG) – AAV-mediated transgene delivery to all branches ot TG
- In vivo mutugenesis of latent HSV
- Specificity – NGS analysis of Off target activity of NV1: Insertion vs Deletion vs NGS analysis of On and Off target activity of HSV1m8
- Endonuclease therapy suppresses viral reactivation
- Viral eradication: critical determinant: 3 of doses before cure occurs
Conclusion
Delivery system is the most important factor
11:05 Nucleic Acid Delivery Systems for RNA Therapy and Gene Editing
Daniel Anderson, Ph.D., Professor, Department of Chemical Engineering, Institute for Medical Engineering & Science, Harvard-MIT Division of Health Sciences & Technology and David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology
High throughput, combinatorial approaches have revolutionized small molecule drug discovery. Here we describe our high throughput methods for developing and characterizing RNA delivery and gene editing systems. Libraries of degradable polymers and lipid-like materials have been synthesized, formulated and screened for their ability to deliver RNA, both in vitro and in vivo. A number of delivery formulations have been developed with in vivo efficacy, and show potential applications for the treatment of genetic diseases, viral infections and cancers.
- Nanoparticulate approaches – repair your DNA while you still use it
- Barriers to Intracellular Delivery – What organs are most amenable to Targeting
- Liver, spleen, bone marrow, kidney
- Intracellular Drug Delivery:
- Modular Pharmaci=ology with siRNA siRNA silences mRNA
- Turning Nuclaic Acids into Drugs: Sequence Selection, Mechanical modification (ligand conjugation), Encapsulation
Materials used for RNA Delivery – increase diversity of materials
- Liquid light material: Combinatorial synthesis of lipid-like materials
- RNA NAnoparticles – Lipid -siRNA-Nanoformulations targeting TTR in the liver of Primates
- Mechanism of ApoE mediated iLNP Delivery [Phil Sharp/Alnylam]
- si delivery to ENdothelium
- Lipid modified Polymers: Short amino polymers – Total Dose 5 siRNA
- Nanoformulation Chemistry: Endothelium in many organs(preferred) vs Hepatocytes
- Immune cells as a target: CD45 or control
- In vivo mediated Homologous Recombination Gene repair: Nanoformulation deliver sgRNA
- In vivo delivery of sgRNA to endothelium wiht nanoparticle: mediated guide RNA delivery to endothilium: DNA Repair and Protein delivery
- Can CRISPR be used to repair a disease gene in vivo – PLASMID encoding Cas9 and GuideRNA + 199nt ss DNA repair template
- in vivo CRISPR rescue repairs defect, restores body weight and stops
- mRNA with nanoparticles: In vivo delivery- EPO Protein: Mean Human EPO
- AAV Only
- AAV +CAs9 – nano – 6% repain is therapeutic
- In vivo mediated Gene Knockout without the Virus
- PCSK( Blood Cholesterol Liver PCSK9 Analysis – 60% gene mutated 65% reduction in CHolesterol — Synthetic system to do gene knockout
11:35 PANEL DISCUSSION: CRISPR/Cas: A Realistic and Practical Look at What the Future Could Hold
Moderator: Bryan R. Cullen, Ph.D., James B. Duke Professor of Molecular Genetics and Microbiology and Director, Center for Virology, Duke University
Participants: Session Speakers
Each speaker will spend a few minutes sharing their viewpoints and experiences on where things stand with using the CRISPR/Cas system for in vivo applications. Attendees will have an opportunity to ask questions and share their opinions.
Discussion
- Ex vivo delivery to Immune cell rather than to the tumor itself
- solid cells therapy needs to reach each cell alternatives are needed
12:05 pm CRISPR/Cas9 for the Screening of the Human Kinome – A Pilot Study in an Aggressive Pediatric Cancer Cell Line
Simone T. Sredni, M.D., Ph.D., Research Associate Professor, Neurological Surgery, Northwestern University Feinberg School of Medicine, Ann and Robert H. Lurie Children’s Hospital of Chicago
The CRISPR-Cas9 system for genome editing is a powerful tool to identify genes involved in vital biological processes. A systematic functional screening of the human kinome has the potential to reveal molecules that are essential for tumor survival, growth, and migration. We will describe our experience using the Invitrogen LentiArray™CRISPR library to mutate 160kinases in a highly malignant pediatric tumor cell line. We will discuss our approach for screening, monitoring of cells lines, and validation.
- LentiArray CRISPR Kinase Library – Bet Test 160 Kinase inhibitors
- MRT – Malignant Rhabdoid Tumors – Children lexx 3 Years old
- Genetic landmark, histology anatomical location: Kidney, Brain, Spine
- Kinase Inhibitors and MRT
- Finding Novel Targets
- Invitrogen – lentiArray CRISPR Library – edit 160 kinase genes – Viral Vector design
- Cas9
- gRNA-Kinase
- Positive Control
- Negative Control
2. Kinome Screening – Impact on Proliferation 160 – only EIGHT were tested – significal=nly impaired cell profiferation
Retransaction and Confirmation of the identified targets
PIM – Leukemia virus induce lymphomas: Proviral – PIM-1,2,3 KO
- Verification of Genome Editing
- PIMs – Proliferation
- PIMs – Senescence – Adult hematological diseases and refractory solid tumors
- PLK4 – Direct Mitosis Regulator – Activated Protein (mRNA) – expression in cytoplasm
- PLK-4 and Cancer: Over-expression vs Deregulation – Colony Formation – colonygenic
- Gene expression – Frozen Tumors – abnormality in children and in adults
- Verification of Gene Editing: Cleavage and deletion
- PLK-4 as a Cancer Drug Target – inhibitor enzymatic – PLK-4 Inhibitor Xenografs
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