An Academic Perspective:

Establishing Standards for Therapeutic 

Genome Editing

Matthew Porteus MD PhD

Department of Pediatrics

Divisions of Stem Cell Transplantation and Regenerative Medicine, Hematology/Oncology and 

Human Gene Therapy

Institute of Stem Cell Biology and Regenerative Medicine

Child Health Research Institute

Stanford University

mporteus@stanford.edu

19 April 2018
Conflicts of Interest

CRISPR Therapeutics: Equity and SAB

Managed through Stanford in accordance with their 

conflict of interests policy.
Take Home Points

1. There are thousands of diseases that affect only a small number of patients and 

establishing a burdensome regulatory path that is not based on scientific risk 

will inhibit genome editing based therapies being developed.

2. Current clinically relevant methods of genome editing using the CRISPR/Cas9 

platform ex vivo are highly specific (more specific than life).

3. Bioinformatic methods identify the relevant off-target sites using clinically 

relevant delivery methods.

4. There are no validated animal models to predict in human genotoxicity for 

genome editing.

• Standard NSG mouse xenograft models do not support most human blood cancers 

(Reinisch et al Nature Medicine (2016) PMID 27213817)

5. Translocations between the on-target break and spontaneous breaks will occur but at a 

frequency that is too low to be detected by current methods.

6. The best test of the safety of genome editing based therapeutics is phase I trials 

for serious diseases with unmet medical needs with reasonable follow-up.
Genotoxicity vs functional toxicity

Peter Marks (head of CBER, FDA): “We don’t want off-target 

events leading to serious adverse events.”

Implication: Off-target changes per se are not serious adverse 

events—only if they lead to functional adverse events.
Nuclease Genotoxicity in HSPCs for an FDA 

Approved HSPC Editing Clinical Trial

4.7

20.2

12.0

1.7

Exon Exon Intergenic Intron Intron
Qualitative Biochemical Understanding of 

Nuclease Specificity

p(Editing) ≈ c1[conc] * c2(Time) * c3(Kd) (on/off) * c4(Kcat) * c5(p(repair fidelity))

Kd ≈ (guideRNA binding energy)(Cas9-DNA binding energy)
Ribonucleoprotein (RNP): Purified Cas9 protein complexed to 

synthetic stabilized guide molecule (gRNA) as the best method to 

engineer cells ex vivo
Importance of 

Delivery and Cell 

Type on Specificity Nature Methods (2017)
Bioinformatic

Oligo capture

In vitro Cas9/gDNA cleavage

Multiple Methods to Identify Potential Off-Target Sites

(ways of creating more lamp posts)
In vitro Methods to Identify R-02 HBB Potential OffTarget Sites

NAR (2013) Nature Methods (2017)

*

*

*

*

*

*
COSMID GUIDE-Seq CIRCLE-Seq Site Sequence Closest Gene Distance (kb) Feature hg19 Location NHEJ Mock

R02 CTTGCCCCACAGGGCAGTAANGG HBB n/a Exon Chr11:5248198-5248220 54.7 (22.0 HR) 0.767

COS1 GS1 CS2 R02_OT1 TCAGCCCCACAGGGCAGTAAGGG GRIN3A 95.004 Intergenic Chr9:104595866-104595888 16.193 0.076

COS2 R02_OT2 CCTCTCCCACAGGGCAGTAAAGG LINC01482 0.034 Intergenic Chr17:66624239-66624261 0.048 0.041

COS3 R02_OT3 TTTTCCCCAAAGGGCAGTAATAG MYO16 n/a Intron Chr13:109818336-109818358 0.012 0.007

COS8 GS2 CS7 R02_OT4 GTGGCCCCACAGGGCAGGAANGG MAGEE2 1.209 Intergenic ChrX:75006240-75006262 0.003 0.005

COS7 GS3 CS4 R02_OT5 GCTGCCCCACAGGGCAGCAANGG FAM101A 3.258 Intergenic Chr12:124803828-124803850 0.153 0.015

GS4 R02_OT6 GATGCCATTCATAGCAGTCANCG C22orf34 225.248 Intergenic Chr22:49582904-49582926 0 0.001

COS23 R02_OT7 CTCGCCCCTCAGGGCAGTAGTGG GREB1 n/a Intron Chr2:11777795-11777817 0.006 0.042

COS9 CS1 R02_OT8 TGTGCCCCACAGAGCACTAANGG LOC101929350 1.3kb Intergenic Chr22:17230606-17230628 0.028 0.064

COS19 CS3 R02_OT9 ATTGCCCCACGGGGCAGTGANGG LOC643339 n/a Intron Chr12:93549185-93549207 0.054 0.016

COS26 CS5 R02_OT10 GTTGCCCCTCAGGACAGTACNGG LOC105370802 374kb Intergenic Chr15:46598112-46598134 n.d. n.d.

CS6 R02_OT11 GAAGCCCTACAGGGCAGCAANGG NRSN1 416kb Intergenic chr6:23709573-23709595 0.024 0.006

COS15 CS8 R02_OT12 ATGGCCCCACAAGGCAGAAANGG IFI27 2.3kb Intergenic Chr14:94585321-94585343 0.013 0.018

CS9 R02_OT13 AGTGCCACACACAGCAGTAANGG DOCK5(H3K27Ac) 110kb Intergenic chr8:24931375-24931397 0.015 0.006

CS10 R02_OT14 TGTGCACCACAGAGCAATAANGG ZNF716 183kb Intergenic chr7:57716460-57716482 0.019 0.04

CS11 R02_OT15 GTTATCCCACAGGACAGTGANGG SFTA3 53kb Intergenic chr14:36889532-36889554 0.055 0.043

All bonafide Off-Target Sites (two) in CD34+ HSPCs 

using RNP were Identified by COSMID

with Ciaran Lee and Gang Bao (Rice University)
”Log-fold improvements will always be 

important in making gene therapy safer”

-Paraphrasing Dr. Chris Baum

(2017 ESGCT Opening Ceremony)
Sickle Cor ection

Of Target 1

Of Target 3

0.01

0.1

1

10

100

percent genome modification

WT

HiFi

Using SCD-CD34s

IDT HiFi SpCas9 Mediates High Level Sickle Gene 

Correction while Reducing Off-Target INDELs by > 1-

log in Sickle Cell Patient Derived CD34+ HSPCs

Vakulskas, Dever, Camarena, Lee, Bao, Behlke

(manuscript under review)
Low Number of Off-Target Sites and Frequency of 

INDELs in Healthy Donor and Patient Derived CD34+ 

HSPCs using RNP Delivery

(Both identified by COSMID--Bioinformatics)

48 other potential off-target sites were analyzed with no evidence of INDELs in 

CD34+ HSPCs modified by RNP

with Ciaran Lee and Gang Bao (Rice University)
Ex vivo Genome Editing is More Precise than Life

• Tremendous genetic diversity among humans to begin with: 

Baseline Variation Per Person: 2.4 million SNVs, 500-600K In/Dels (355 Exonic, 91 Frameshift), 

~3000 structural variants (i.e. Dewey et al 2014 JAMA)

• Tremendous ongoing genetic diversity within each person

10-20 new mutations per every cell division

1.00E+05

1.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

1.00E+11

1.00E+12

1.00E+13

1.00E+14

1.00E+15

1 2 3 4 5 6 7

1015

1014

1013

1012

1011

1010

109

108

107

106

10

Number of New Mutations

5

Assumes high fidelity of DSB Repair: 90% (9 out of 10 breaks are repaired precisely)
Proposed Standards

1. Analysis of potential off-target INDELs should be performed at all 

potential off-target sites in which there are 3 or fewer mismatches 

(excluding nucleotide 20) and no PAM mismatches identified bioinformatically using the delivery method and cell type that is part of the 

target product profile (TPP).

• Other techniques can supplement but not replace this analysis of 

potential off-target sites.

2. If on-target site or off-target sites with known INDELs are not associated 

with cancers (for the tissue type that might be modified), then no 

further genotoxicity/tumorigenicity studies need to be performed.

3. If on-target site or off-target sites with known INDELs are associated 

with cancers (for the tissue type that might be modified), then 

thoughtful functional assays for genotoxicity/tumorgenicity should be 

performed.

4. Samples from patients treated with genome edited therapeutics should 

be archived (15 years?) for analysis if genotoxicity/tumors occur. 
Porteus Lab (current)

Joab Camarena

Carsten Charlesworth

Kyle Cromer PhD

Daniel Dever PhD

Natalia Gomez-Ospina MD, PhD

Kazuya Ikeda PhD

Vienna Kuhn

Sruthi Mantri

Renata Martin

Nathalie Mostrel

Mara Pavel-Dinu PhD

Samantha Scharenberg

Camille Sindhu PhD

Wai Srifa

Sriram Vaidyanathan PhD

Volker Wiebking MD

Ginger Exley

Cita Nicolas

Loan Nguyen 

Annalisa Lattanzi PhD

Funding: 

NIH/NHLBI/NIAID, CIRM, Amon Carter Foundation, Laurie 

Kraus Lacob Faculty Scholar Fund, Binns Family Cord 

Blood Research Program. Chan-Zuckerberg Initiative, 

Sutardja Foundation, Taube Foundation, NORD, Thrasher 

Foundation

Division of Pediatric SCTRM

Maria-Grazia Roncarolo

Sandeep Soni

Rajni Agarwal

Ken Weinberg

Rosa Bacchetta

Ami Shah

Robby Parkman

Katja Weinacht

Agnieszka Czechowicz

Alice Bertaina

Julia Chu

Melissa Mavers

Collaborators

Anu Narla

Jennifer Andrews

Michael Jeng

Bert Glader

Mohan Narla/Lionel Blanc

Rachel Lynn/Crystal Mackall

Nobuko Uchida

Ann Tsukamoto (BOCo)

Ayal Hendel (Bar-Ilan Univ)

Rasmus Bak (Aarhus Univ)

Andreas Reinisch/Ravi Majeti

Michael Cleary

Greg Gurtner

Joe Wu

Calvin Kuo/Tushar

Desai/Jayakar Nayak

Mark Behlke (IDT)

Chris Vakulskas (IDT)

Suk See DeRavin (NIH)

Harry Malech (NIH)

Gang Bao (Rice)

Ciaran Lee (Rice)

Laboratory of Cell and

Gene Medicine (LCGM)

David DiGiusto

Chy-Anh Tran

Neehar Bhatia

Ulrike Jung

Helen Segal

Abla Bakir

Premanjali Lahiri

Mauricio Umana

Rashi Srivastava

Thank You

(for your attention and the opportunity to present our work)