amyotrophic lateral sclerosis and stem cell therapy

2019

Genome Editing Technologies and Neuromuscular Disease

Nicolas N. Madigan, MB BCh BAO, PhD

2019

Financial Disclosure

Relevant Financial Relationship(s)

None

Off Label Usage

None

2019

Learning Objectives

o Understand the mechanisms of genome editing technologies

o Learn how gene editing is being used to develop new therapies for neuromuscular disease

o Consider the translational challenges for genome editing technologies

2019

Genome Editing• Refers to a process of making precise and permanent changes

in the genetic code of cells and organisms

• 3 stepso Targeting a nuclease enzymeo Cutting the genomic DNAo Inducing DNA repair change

• Applications in Neuromuscular Diseaseo Repairing genetic mutations (delete, edit, add genetic elements) o Activating correct - or inactivating incorrect - gene expression, in a therapeutic manner

McGovern Institute for Brain Research, MITwww.pbs.org/wgbh/nova/

2019

single-guide RNAZFN: Zinc Finger NucleaseTALEN: Transcription Activator-Like Effector Nuclease

Genome Editing SystemsProtein-Guided RNA-Guided

Heidenreich M. and Zhang F. Nat Rev Neurosci 2016; 17:36-44

Accurate Targeting

Frame-shift knockout

Targeted cut

Repair

Non-homologous end joining

Homology-directed repair

Knock-in or gene correction

Nuclease

2019

Multiplexing

Heidenreich M and Zhang F. Nat Rev Neurosci 2016; 17:36-44

NHEJ

2019

Marraffini LA. Nature 2015; 526: 55-61

Transcription of Full Length crRNA

by RNase III

CRISPR - Array

ClusteredRegularlyInterspacedPalindromic Repeat

CRISPR-Cas9 is an Adaptive Immunity System

CRISPR-associated (cas) operon

Adaptation

Maturation

Interference

Cas1 Cas2Cas9 Csn2

2019

Class 1 and Class 2 CRISPR Systems

Mohanruju M. et al. Science 2016; 353: aad5147

AncestralSystem

Type I

Type III

Type II – Cas9Type V – Cas12a (Cpf1)

Type VI - Cas13 (C2c2)

EFFECTOR MODULEADAPTATION

MODULE

2019

Cas9 Diversity Among Bacterial Species

Barrangou R. and Doudna JA . Nature Biotechnology 2016; 34: 933-941

2019

CRISPR-Cas Systems in Use

Cas9• Class 2, type II

• sgRNA dsDNA

• Cas9 nuclease / helicase

• Blunt end

Cas12a (Cpf1)• Class 2, type V

• Single crRNA ss + dsDNA

• Cas12a nuclease / helicase

• Sticky end overhangs

Cas13a (C2c2)• Class 2, type VI

• crRNA ssRNA

• HEPN nuclease /RNAase

Knott et at, Science 2018 :361:866-869

2019

sgRNA-Cas9 Complex Surveillance

Jinek M. et al. Science 2012; 337: 816-821

sgRNA

Cas9 GenomicDNA

McGovern Institute for Brain Research, MIT

20 nt of your choice

RuvC

HNH

Cas9 proteins have dual helicase and nuclease functionality

2019


Madigan NN, et al Neurology. 2017 Oct 17;89(16):1739-1748 McGovern Institute for Brain Research, MIT

TargetApproach

• Cas9 proteins form ribonucleoprotein (RNP) complexes with sgRNA molecules

• Number of DNA regions that can be sampled depends upon RNP concentration

2019



TargetEngagement

protospacer

• RNPs will bind DNA wherever they encounter a protospacer adjacent motif (PAM)

• Assessment of the first 5 – 6 nucleotides (“seed region”) ? Full complementarity

Madigan NN, et al Neurology. 2017 Oct 17;89(16):1739-1748

2019

sgRNA-Cas9 Cutting


TargetCutting

• Double-stranded cut = 3 nucleotides upstream from the PAM

RuvC

HNH

Madigan NN, et al Neurology. 2017 Oct 17;89(16):1739-1748

2019

PAM sequence availability can affect edit precision

• spCas9 (NGG > NAG)o 5.2 % genomeo Every 42 base pairs (on average)o > 161 million occurrences

• Hierarchy of favored NTs• Staph aureus

o NNGRRT

• S. thermophileso NNAGAA and NGGNG

• N. meningiditiso NNNNGATT

• Cas12A/Cpf1o TTTV (A/C/G)

2019

“On” Target Effects• Easy to measure• A ‘good’ reagent will cut > 50 % of alleles (indel)

• Gene integration by HDR: < 5 – 15 % of alleleso Cell cycle dependent (S/G2) o Varies with cell quiescenceo Can employ homology arms of different lengths and mechanisms

• MMEJ - <50 bp overlap vs ‘long arm’ HDR (> 0.5 – 2 kb)

• Always be a mixture of outcomes: proportions of genomes with integration, proportions with indels, and those remaining unmodified.

2019

“On” Target Effects

• Cutting efficiency largely depends on ‘dose’ of the editing reagent o Efficient delivery of the reagento How long reagents are produced from their vector templateo Reagent degradation

• Variables intrinsic to the guide sequence (NT composition)• Variables intrinsic to the target

o Chromatin accessibility, methylation state

2019

“Off” Target Effects• Occur with all gene editing technologies• Engaged & cut DNA where you did not intend to• Extremely difficult to measure• Paramount importance for patient safety

• Improved by comprehensive strategies:o On-target site selection algorismso Off-target computer prediction models for a given guide sequenceo Detection by advanced DNA sequencing technologieso Reducing reagent dosage (i.e. the time it is available, concentration)

2019

REAGENTDELIVERY

Yes

2019

Broadening CRISPR-Cas Functionality

Single stranded cuts• Inactivation of individual nuclease

domains• Amino acid substitutions

• D10A (RuvC inactivation)• H840A (HNH inactivation

2 Nickases used in tandem / multiplex• Produces stick-end overhangs• Ease integration of DNA fragments

with corresponding ends

• Single strand cuts may force particular repair pathways (BE)

2019

Broadening CRISPR-Cas Functionality


KRAB repressor

VP16, VP64‘SunTag’ GCN4 peptide activators

Drug induced:DOXYRUBICIN

P300 histone acetylmethylases

LSD1 histoneTET1 demethylases

Dynamic Chromatin ImagingCas9 binding kineticsDNA detection technologies

Optogenetic domains[light induced transcriptional control]

Heidenreich M and Zhang F. Nat Rev Neurosci 2016; 17:36-44

“dead” Cas9

Targeting capabilitiesWithout cutting

Delivery of Effector domains

2019

CRISPR “Base-Editing” [BE] Systems• 4 Precision BE systems• Combine:

• dCas9 targeting+/- ‘nickase’ activity

• CBE or ABE isoform• UGI

• High rates of off-target and indel mutations

• Target “window” bp range

CBE: cytidine base editor – C:G bpA:T bpABE: adenosine base editor – A:T bp C:G bp

Young CS, et al Physiology 2019:; 34; 341-353 Hess GT, et al Molecular Cell 2017; 68: 26-43Barrangou R. and Doudna JA . Nature Biotechnology 2016; 34: 933-941

2019

Knott et al. Science 2018 :361:866-869

Ability to edit across gene expression paradigm

2019

Gene Editing for Neuromuscular Disease

In Vivo Gene Editing Ex Vivo Gene Editing

2019

CRISPR-Cas9 systems in Clinical Trials

• 21 Active Clinical Trials (recruiting / not yet recruiting) o USA (8), China, France, Germany

• Ex vivo trials• Majority in hematology and oncology using CAR-T cells

engineered with CRISPR-Cas gene knockdown to enhance killing of malignant cellso PD-1, CD7 modifications

• No current neuromuscular disease trials

www.clinicaltrials.gov

2019

Ex vivo Gene Editing• Gene-editing reagents are expressed and act only within the

extracted and retransplanted cell population• SAFETY and EFFICIENCY advantages

• Setting and reaching targets of accuracy when cutting and repairing DNA in cells

• Predict mutational outcomes at on and off target locations

• Control the cell cycle for enhancing HDR efficiency

• Immune benefits of autologous transplantation

2019

Paradigm for ex vivo Gene Editing

Ex Vivo Vectors

NHEJ

HDR

iPSCs MSCs T-Cells Schwann Cells- Factor Secretion

for trophic support- Immunomodulation

- Exosomes

- Seek and Destroy- Immune-Response

Modulation

- Peripheral Neuropathy

- Spinal cord injury

- Disease Modeling- Implantation as corrected,

differentiatednerve, glial or muscle cells

-

2019

In vivo Gene Editing

• Gene-editing reagents are delivered directly to cells in the CNS, PNS or muscle

• Potential gene editing applications currently are focused on monogenic disorders with a clear, individual target and correction mechanism.

• Advantages: opportunity to directly correct pathologic cause; versatility of platform to address diversity of cause mechanisms

• Disadvantages: permanence of unintended consequences; inability for cell selection or sorting; no longer in an immune-privileged environment

• Technical challenges of reagent delivery, efficacy and safety in the human body as a whole

2019

Paradigm for in vivo Gene Editing

In Vivo Vectors

Non viral AAV

1) Cutting• NHEJ gene knockdown• Exon excision• Repeat expansion

excision• Exon reframing• Splice site modification

NHEJ

2) Cutting + Recombination• Gene correction:

• Point mutation with Oligonucleotide template (MMEJ)

• Larger sequence exchange by HDR

3) dCas• Transcriptional

control• Methylation control• Base pair exchange

2019

Neuromuscular Disorders and

CRISPR-Cas – Preclinical studies

• 3 Main Disease Categories:• Congenital Muscle Disease

o Duchenne Muscular Dystrophyo Limb Girdle Muscular Dystrophies Type 2B (Dysferlin), Type 2D (α-sarcoglycan)o Fascioscapulohumeral Muscular Dystrophy (FSHD)o Congenital muscular dystrophy type 1A (MDC1A) – LAMA2 / Merosin deficiency

• Familial Amyotrophic Lateral Sclerosiso Superoxide dismutase (SOD)o C9ORF72 GGGGCC hexanucleotide repeat expansion

2019

Neuromuscular Disorders and

CRISPR-Cas – Preclinical studies

• Repeat Expansion Disorderso Myotonic dystrophy types 1 and 2 (CTG repeat in DMPK, CCTG repeat in CNBP)o Huntington Disease (CAG repeat in HTT)o Spinocerebellar ataxia type 2 (CAG repeat in ATXN2)o Friedrich ataxia (GAA repeat in FXN)o Fragile X (CGG repeat in FMR1)

2019

DMD Gene Editing• “Efforts to use CRISPR-Cas9 mediated genome editing to correct

muscular dystrophy are setting the pace for clinical implementation.”

Babacic H, et al. PLoS ONE 2019: 14(2) e0212198


DMD: 26/42 studies

2019

DMD Gene Editing

• Strategies: depend upon the model used, but majority of studies:o EXON SKIPPING / EXON EXCISION by NHEJ repair = REFRAMING Strategies

READING FRAME RESTORATION

Convert DMD BMD genotype

Babacic H, et al. PLoS ONE 2019: 14(2) e0212198Young CS et al Physiology 2019: 34:341-353

*

*

*

“Stop” removal

“Stop” or duplication removal

mdx mouse∆Exon 23(46% of studues)

‘humanized’ mdx mouseImmortalized human muscleHuman iPSC∆Exon 50 canine

2019

DMD CRISPR-Cas Reagent Delivery

Viral: 55%Non Viral:45%

19/26 studies were in vivo in animal models

• Included ex vivo modification and transplantation

• 3/26 studies attempted HDR exon replacement after excision

Babacic H, et al. PLoS ONE 2019: 14(2) e0212198Lim KRQ et al. J Pers Med 2018; 8 (38): 1-20.

2019

DMD in vivo Gene Editing: Double cut• mdx mouse model –

o Premature stop codon in Exon 23

Long CH et al. Science 2016; 351:400-403 Nelson CE et al. Science 2016; 351:403-407

2019

DMD: Double cut +/- HDRo Mouse model Exon 53 premature stop codon

Muscle specific promotor, spCas9 or saCas9

Exon 51 Exon 54 NHEJ HDR repair

Partial in-frameExon 23 deletion

Bengtsson NE et al Nat Commun 2018;14; 14454

2 AAV viruses needed for SpCas9 system

1 AAV virus needed for SaCas9 systemor

2019

Bengtsson NE et al Nat Commun 2018;14; 14454

DMD Double cut +/- HDR

Systemic delivery of high dose virus (1-10 x 1012 vg) restored widespread, but variable dystrophin expression in skeletal (<10 to

>50% fibers) and cardiac muscle (up to 34% fibers)

2019

Single cut at Splice Site: DMD Canine Model

• ∆Exon50-MD Canine Model: affects the reading frames of exons 51 (premature stop) – 79; recapitulates muscle weakness, atrophy and fibrosis of human disease.

• Correctable both by reframing of exon 51 and by exon 51 skipping

Amoasii L et. al. 2018 Science; 362: 86-91

ppp

• Single sgRNA targeting region adjacent to the exon 51 splice acceptor site• Highly conserved between canines and humans (one nucleotide difference)

2019

Single cut at Splice Site: DMD Canine Model

• ∆Exon50-MD Canine Model: affects the reading frames of exons 51 (premature stop) – 79; recapitulates muscle weakness, atrophy and fibrosis of human disease.

• Correctable both by reframing of exon 51 and by exon 51 skipping


ppp

2019

• Systemic delivery of AAV9-sgRNA-51

• Dose dependency of dystrophin expressiono 1 x 1014vg/kg > 2 x 1013vg/kgo Cranial Tibialiso Semitendinosiso Bicepso Tricepso Diaphragmo Heart o Tongue

Single cut DMD

canine model

Large animal study paving the way forward to human clinical trials


2019

DMD Base Editing (mouse)

o D10A NickaseCas9 with ABE7.10

o Aim: Convert TAG stop CAG Glnin exon 20

o Substitution frequencies up to of 70% in vitro

o AAV injection into TA muscle

o 3.3% base editing efficiency

o 17% dystrophin + fibers

Ryu et al. Science 2016; 351:403-407

2019

DMD editing outcome highlights• Most studies:

o High rates of exon excision efficiency, vector dependent: 60 – 90 % of transcriptso Variable restoration of dystrophin expression

• Western blot• Immunohistochemistry (percent + muscle fibers)

o Typically ranging from 0% to 50% over control

o HDR rates were about ~<5% in vivo

• Much work into model development• Improvements over time with optimization of targeting

parameters• Sites targeted, efficiencies of reagent delivery

Babacic H et al. PLoS ONE 2019: 14(2) e0212198Lim KRQ et al. J Pers Med 2018; 8 (38): 1-20.

2019

dCas9 transcriptional upregulation of

Utrophin in vitro DMD model• Myoblasts from affected DMD patient (∆Exons 45-52)• dCas9 with 10 tandem repeats of the transcriptional activator

VP16 • Strategy to compensate for dystrophin loss by upregulating

utrophin expression• dCas9 activator guided by sgRNAs to the utrophin A or utrophin

B promotor regions (or their combinations)o Demonstrated an 1.7 to 6.9 fold increase in utrophin protein in vitro using individual guideso Higher with combination strategies targeting B

Wojtal D et al. Am J Hum Genet 2016; 98:90-101.

2019

dCas9 transcriptional upregulation for

Laminin α2 (MDC1A) deficiency

• dy2j/dy2j mouse model with LAMA2 splice site mutation causing exon skipping and N-term protein truncation

• Strategy to compensate for laminin-α2 loss by upregulating laminin-α1 expression

• dCas9 flanked by the transcriptional activator VP64 flanking to target promoter

• Viral sgRNA array delivery for targeting promotor at multiple sites

Kemaladewi DU et al . Nature 2019; 572:125-130

LAMA1 Promoter Region

2019

• Improved laminin-α1 expression• Improved the disease phenotype

over 5 weeks compared to sham treated animals

Kemaladewi DU et al . Nature 2019; 572:125-130

2019

FSHD: Transcriptional Downregulation

• Targeting D4Z4 microsatellite repeat de-repression o D4Z4 encoded RNA

• Misexpression of full length DUX4 retrogene within distal-most repeat o In the presence of the

permissive haplotype allele

• sgRNA and dCas9-KRAB repressor targeted to Dux4 promotor region or exon 1 by lentiviral delivery

• Reduced DUX4 expression by 45% in cultured FSHD patient myocytes

• Reduced downstream expression of secondary germline genes (TRIM43, ZSCAN4, MBD3L2) by 35-60%

Himeda CL et al. Mol Ther 2016; 24:527-535

DUX4 promotor Or Exon 1

2019

ALS: SOD mouse knock down

• SaCas9 – AAV to Exon 2 of the SOD gene in the G93A-SOD1 mouse by facial vein injection

• Single guide system

• Reduced SOD1 protein by 3 fold in the spinal cord, despite low indelformation rates (<1 % in SOD transgenes)

Gaj T et al. Sci Adv 2017; 3: eaar3952 1-10

2019

Delayed disease onset Improved survival

Improved motor performance

Improved maintenance of animal weight


AAV9-SaCas9-hSOD1

wt

AAV9-eGFP / mRosa26

AAV9-SaCas9-hSOD1

2019

Improved numbers of motor neurons at end stage disease

Emphasized concept that high rates of recovered protein expression may not be needed to positively influence the phenotype


2019

Long C et al. JAMA Neurol 2016; 73:1349-55

ALS/FTD

MyotonicDystrophy

Gene editing for repeat expansion

2019

DMPK- CTG expansion removalSelection of 3 effective sgRNA, 3’UTR region

• Effective removal of short hairpin repeats (5) (67% cells) and long (540-610) repeats (30%) in DM500 (mouse) and DM11 (human) myoblast lines

• Cleared cells of ribonuclear foci

• Without influencing downstream SIX5 expression, or myogenic differentiation

Van Agtmaal EL et al. Mol Ther 2017; 24: 24-43

• Guide efficiency determined by Sanger sequencing and ‘TIDE’ analysis (frequency and position of indels)

• Prediction software for off-target binding propensity

2019

dCas9 transcriptional downregulation of

microsatellite repeats by steric inhibition• Used dCas9 targeting

sgRNA to repeat expansion in in vitro models of DM1, DM2 and C9ORF72 repeats

• Targeting plasmids with 12-960 repeats

• sgRNA with constrained PAM (not ideal NGG)

• Each of the 3 nucleotide phases

• Multiple RNPs binding on repeats, blocking polymerase

Pinto BS et al. Mol Cell 2017;68:479-490

2019

dCas9 transcriptional downregulation of

microsatellite repeats

• Reduced repeat RNA expressiono Length dependent (longer > shorter

due to increased binding)o Targeting the non-template strand o PAM dependent

• Reduced repeat associatednon-ATG (RAN) translation

• Reduced pathologic RNA foci in myoblasts

• Reduced prevalence of myotonic discharges in mice

Pinto BS et al. Mol Cell 2017;68:479-490

2019

“RCas9” transcriptional degradation of

microsatellite repeats in DM1

• Utilized a re-purposed Cas9 to bind RNA repeats directly

• Fusion to PIN RNA endonuclease for repeat degradation

• Plasmid systemo 105 CTG repeats (DM1)o 200 CCTC repeats (DM2)o 80 CAG repeats (HD)

• Effective RNA binding and cleavage

• Reduced pathologic RNA foci in target cells

Batra R et al. Cell 2017; 170: 889-912

Produced truncated versions of the RCas9-PIN endonuclease for lentiviral packaging and reduction of repeat expression in DM1 patient myotubes.

2019

Insertion of PolyA tail before 3’UTR repeat

• Excision of repeat region in DM1 patient iPSC associated with high frequency of inversion

• Different approach:• Integrate a polyA signal afterExon 15 and before the 3’ UTR expansion to complete the transcript• Lead to the elimination of intra-nuclear CUG RNA foci and reversal of abnormal splicing in iPSC and iPSC differentiated to cardiomyocytes neural stem cells

Wang Y et al. Mol Ther 2018; 26:2617-2630.

2019

Fragile-X FMR1 targeted demethylation

• In FXS, FMR1 gene silencing associates with hypermethylation of the 5’UTR CGG expansion

• Causes heterochromatin formation at the FMR1promotor and subsequent gene silencing

• Approach: use dCas9 fused to Tet1, a demethylase.

• Delivered to FX iPSC by lentivirus

Liu XS et al. Cell 2018; 172: 979-992

2019

Fragile-X FMR1 targeted demethylation

• Restored FMR1 expression to 73%-90% of wild type in FX-iPSC, and reduced local methylation to 4%.

• Edited iPSC neurons had rescued mean firing rates

• Maintained FMR1 expression in vivo upon engraftment into mouse brain

• Demethylation of CGG repeat in post mitotic FXS neurons reactivated FMR1

Liu XS et al. Cell 2018; 172: 979-992

2019

Strategies for repeat expansion disorders

Raaijmakers RHL et al. Int J Mol Sci. 2019; 20: 3689. Liu XS et al. Cell 2018; 172: 979-992

e. Demethylation

2019

Nelson CE et al. Nat Medicine 2019; 351:403-407

Long term studies: DMD Editing

• AAV vector was persistent in cardiac tissue between 8 weeks and 1 year, but significantly lost in skeletal muscle

• Expression of the saCas9 mRNA and gRNA virtually absent after 6 months, ? Promoter silencing.

Long term dystrophin expression

2019


Unintended sequelae

2019

Host response to bacteria-derived proteins

• Antibodies against saCas9 detected in treated adult mice.• T-cells from saCas9 adult mice could be readily restimulated to produce

INF-γ.• Neither response was seen if the mice were treated as neonates.

Analogous to humans:• S. Pyogenes, S. Aureus are extremely common infections in humans• 22 human cord blood donors

o SaCas9 and SpCas9 antibodies were found in 78% and 58% of donorso Anti SaCas9 and Anti SpCas9 T-cells in 78% and 67% of donors

• Significant portions of people with humoral and cell-mediated adaptive immunity to CRISPR-Cas reagents.

Nelson CE et al. Nat Medicine 2019; 351:403-407 Charlesworth CT et al, Nat. Medicine 2019; 25: 249-254

2019

If AAV is to be the preferred vector

for nerve and muscle…

• “Although the safety of AAVs as a gene-delivery vehicle has been shown preclinically and through over 100 clinical trials, the potential genotoxicity of the combination of AAV and CRISPR requires further characterization of unintended genome-editing events and AAV genome integrations.”


2019

Further preclinical considerations for AAV

• Optimal dosing must counter-balance transduction and editing efficiency with immune response generation

• AAV carrying capacity is limited, frequently studies need to employ dual vector strategies = 2 fold dilution

• Inability to redose AAV will reduce editing efficacy• 30 – 70% population may have a pre-existing immunity to the

AAV, based on sero-prevalance studies• Potentials for AAV integration and therefore to sustained Cas9

expression and activity, or sustained episomal expressiono Long term auto-immune response, and increasing off target activity

2019

Further preclinical considerations for AAV

• Identifying outcome measures in humanso Overcoming barriers of sheer body mass in reagent delivery and tissue targetingo Safety measures in on – off targeting by deep sequencing in vast range of tissue type o How you look is important: sequencing methods can miss large genetic deletions (intronic)o Tissue distribution of the vector o Functional measures may be highly specific to individual patients and disease genotypes

• Exploring means to adapt the immune system to treatmento Immunosuppression vs induction of tolerance vs viral capsid modification ?o Development of self-limiting or self cleaving vector systems / integrated kill-switches / co-

delivery of gRNA against Cas9o Development of non viral or other transient vectors

2019

Conclusions and Future Directions• CRISPR-Cas – is an accessible and adaptable platform, by which specific

tools can be applied to a wide variety of tasks in altering and regulating the genome

• Work in identifying neuromuscular disease genotypes and pathologic genetic mechanisms will soon pay dividends in patients in clinical translation of repair strategies

• Highlighted several examples of how DNA targeting and editing may be accomplished in pre-clinical studies for neuromuscular diseases

• Anticipate an acceleration of RNA-guided RNA editing in this fieldo Added benefits of not inducing permanent changes to the genomeo Building on other RNA silencing technologies (ASOs) in clinical use

• Work is rapidly advancing in improving nuclease binding and modification accuracy, and in developing safer, more efficient vector delivery systemso Robust methods for predicting and measuring modification, to reduce off target events and achieve the

desired editing outcomes

2019

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amyotrophic lateral sclerosis and stem cell therapy

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