Slides from the Genome editing & gene therapy Impact.tech seminar, hosted by Eric Kelsic on June 11th, 2019.
The seminar covers the experiments and inventions that led to the development of genome editing technologies. These inventions were derived from life itself: isolated from natural organisms and adapted for scientific and therapeutic goals. You will learn the history of how genome engineering tools, including CRISPR, and delivery technology, including AAV capsids, were created in their modern form. The seminar explores how genome editing and gene therapy technologies are giving individuals control over their own genomes, focusing on the treatment of genetic diseases. It will describe major companies and emerging trends in the gene therapy industry. Finally, the seminar will discuss how and where new discoveries, including accelerated algorithms for genetic engineering, will lead us in the near and distant future.
Eric Kelsic, PhD, is the founder and CEO of Dyno Therapeutics, a VC-backed biotech located in Cambridge, Massachusetts. Dyno is leading a machine learning revolution to develop enhanced capsid proteins that enable new gene and genome editing therapies. Eric co-developed the technology underlying Dyno’s machine-guided protein engineering platform as a Staff Scientist in George Church’s lab at the Wyss Institute of Harvard Medical School. He holds a PhD in Systems Biology from Harvard University and a BS in Physics from Caltech.
2. Genome Editing
and Gene Therapy
Eric Kelsic
CEO Dyno Therapeutics
Cambridge, MA
Impact.Tech Seminar
San Francisco, June 11, 2019
1
3. Outline of our seminar
I: Genome editing new tools
II: AAV gene therapy new cures
III: Genetic engineering the actual
IV: What next? the possible
2
4. Self introduction
3
Systems Biology PhD
w/ Roy Kishony
@ Harvard
Physics BS
@ Caltech
Postdoc
w/ George Church
@ Wyss Institute
CEO Dyno
Therapeutics
Puzzles
Programming Startups
AAV
5. The journey toward modern medicine
4
Galen’s surgeries based on animal
dissection, dissection of human body
is taboo
Hippocratic Oath
prohibits euthanasia and abortion,
prohibits surgery for kidney stones
(lithotomy), prohibits divulging of
medical secrets
Renaissance: human
dissections and modern
anatomy 1800
1600
400 BCE
200 CE
Pasteur, Koch,
Cell & Germ Theory
1900
Blood
transfusions
DNA
structure
2000
Human
genome
6. Millions of patients have untreatable genetic diseases
5
•10,000+ known
monogenetic diseases
•1% of births worldwide
•Causing 20-30% of infant
deaths in US
Not treatable with current small
molecule or biologic drugs
WHO, Berry et al. 1987
14. Bioprospecting: treasures in the biosphere
• Nature has invented
~1e42 different protein
sequences
• 1e42 upper bound
• 1e30 bacteria in the
world
• 1e3 genes/cell
• Each gene changes each
year for 1e9 years
13
see Dryden, J. R. Soc. Interface, 2008 David Goodsell
16. CRISPR-Cas is a bacterial immune system
using an RNA-guided endonuclease
15
Rath, Biochimie 2015
Spacer sequences from
invading phage are stored in
the CRISPR module
RNA guided complex identifies and cleaves
protospacer sequences from invading
phages, interfering with phage infection
17. There are a wide variety of
biochemical mechanisms
for CRISPR interference
16
Rath, Biochimie 2015
18. Cas9 sgRNA enables simple programmed genome editing
17
Jinek, Science 2012; see also Gasiunas, PNAS 2012
20. Adapting Cas9 for human genome editing – adding a
nuclear localization sequence (NLS)
19
Mali, Science 2013
Cong, Science 2013
Jinek, eLife 2013
21. Applications of Cas9 for basic
research
20
Wang, Science 2014; Shalem, Science 2014
Multiplexed programmable genome
perturbations perhaps the most
impactful initial application of Cas9:
• Knockout
• Activation
• Repression
• Epigenetics…
22. How will human therapeutics be developed?
21
Pro Con
Germ cells Mutations are inherited Unknown risks of off-target
mutations, ethics
Somatic cells Mutations are not
inherited
Challenge of delivery
ex vivo Enables screening for
positives and removal of
negatives
Complicated manufacturing
and transplantation
procedures
in vivo Easier to manufacture and
administer
Challenge of low efficiency
editing, dangers of off-
target editing
Question#1Question#2
✗
✓
34. Frontiers in base editing
• Precision of edits
• Reduced off-target edits
• In vivo delivery
• Reduced size (smaller Cas) for use in AAV
• Or viruses/capsids with larger capacity
33
35. Summary
• Excitement around genome editing driven by potential to understand
and treat genetic diseases with high unmet need
• Cas9 is an RNA-guided endonuclease enabling programmable genome
cutting, nicking, binding, deletion, insertion, substitution, …
• Base editing enables precision substitutions
• Broad consensus around the benefits of somatic cell genome editing
therapies, uncertainty around when germline editing will be acceptable
• Genome editing therapeutics can be ex vivo cell therapy or in vivo gene
therapy
34
37. Why gene therapy?
36
MOST DRUGS ARE INHIBITORS MANY GENETIC DISEASE ARE
CAUSED BY LACK OF THE PROTEIN,
SO THERE’S NOTHING TO INHIBIT
GENETIC MALFUNCTIONS ARE
CHALLENGING TO TREAT USING
SMALL MOLECULES
38. Principles of in vivo gene therapy
37
Gene Therapy DNA Payload
=
Delivery
+
Replace or fix the
malfunctioning gene
39. Why use viral capsids?
38
LIPID NANOPARTICLES ARE
MATERIALS
CAPSIDS ARE MACHINES
• Flexible membranes
• Variable stickiness, reacts to
chemical properties
• Piggyback on endogenous
cellular machines
• Combinatorial chemical
synthesis
• Strong shell
• Senses and responds to
environmental signals
• Manipulates endogenous
cellular machines
• Genetic encoding enables
multiplexing
40. Gene therapy: early years
39
Friedmann & Roblin, first
proposal for gene therapy
1972
1980
1990
First attempts to treat
humans in clinical trials
For ADA-SCID, ex vivo via
lentivirus delivery
Development of viral delivery
vectors: Adenovirus, Lentivirus
Trials begin for in vivo gene
delivery using Adenovirus
2000
42. Adeno-Associated Virus is a small non-replicative
virus (dependovirus genus)
41
Kotterman, Nat. Rev. Gen. 2014
43. Adapting AAV for gene delivery
42
Kotterman, Nat. Rev. Gen. 2014
44. Advances in AAV therapeutic development
43
Dunbar, Science 2018
45. Immune suppression for gene therapy
• Prednisone
• Prevents inflammation
• Prevents immune system from
eliminating cells with viral
capsids
• Transient dosing schedule
concurrent with AAV
administration
44
46. Gene therapy for Leber’s Congenital Amaurosis (LCA)
• Payload: RPE65 (Retinoid
Isomerohydrolase, makes
retinol)
• Results in loss of vision then
blindness during childhood
• Prevalence: 1 in 200,000
• Subretinal AAV2 delivery
• Dose: 1.5e11vg/eye in 0.3mL
• Prednisone 3 days prior and
7 days after injection
• Spark Therapeutics
45
47. Broadening the potential of AAV
with new serotypes
46
Gao, J Virol. 2004DiMattia, J Virol. 2012
AAV2, 4, 8 & 9
49. Gene therapy for Spinal Muscular Atrophy (SMA type 1)
• Payload: SMN1 (Survival motor
neuron protein)
• Results in deterioration of motor
neurons, loss of mobility and then
death by 2 years
• Prevalence: 1 in 10,000
• Intravenous AAV9 delivery, self-
complementary vector
• Dose: 2e14vg/kg in 10-20mL/kg
• Prednisone as needed to control
immune response
• Avexis
48
50. Scaling up to meet manufacturing challenges
49
Vigene
Baculovirus for
large scale production
Other systems using
herpesvirus transduction,
suspension cells,
…
51. Clinical results
• The Gene Doctors, LCA treatment (39:17-43:14)
• https://www.pbs.org/video/the-gene-doctors-i1dd2h/
• Spinal Muscular Atrophy Treatment at Nationwide Children's -- Brett & Paige
• https://www.youtube.com/watch?v=3FyainVRdRc
• Gene Therapy for SMA Type 1: Evelyn's Story
• https://www.youtube.com/watch?v=yRrqbvUv6gQ
50
56. Gene therapy frontiers: payloads
• Increased expression
• Control of dynamics
• Reduced size for large genes
• Dual and multi-payloads
• Combinatorial therapies
55
57. Gene therapy frontiers: capsids
• Not all cells and organs are reachable
• Limitations of cost and manufacturing capacity
56
58. Summary
• In vivo gene therapy delivers a genetic payload into your body’s cells
• Viral capsids are efficient machines for in vivo delivery
• AAV capsids enables in vivo disease treatment
• LCA, SMA, DMD, and many more…
• Gene therapy field has consolidated around AAV technology
• Early gene therapies are a roadmap for translation of in vivo genome
editing therapies
• With innovations on payloads and delivery, curing all genetic diseases is
within reach in our lifetime
57
60. Genetic engineering: how to develop new therapies for
treating disease as quickly as possible?
• Domesticating and improving our genetic natural resources
59
61. How can we make AAV capsids better for gene therapy?
60
Efficient delivery to target cells and tissues
Reduced off-target delivery
Robust evasion of pre-existing immunity
Increased packaging size
High-titer manufacturing capabilities
Natural
capsids
✓
✗
✗
✗
✓
Ideal
✓
✓
✓
✓
✓
62. AAV sequence space is vast and mostly unexplored
Explored:
1-4 mutations
between serotypes:
100-300 mutations
AAV2
AAV5
AAV9
AAV8
61
63. Genetic engineering: recombinant DNA technology
Tools for DNA manipulation
• 1972: plasmid cloning to propagate DNA in bacteria
• 1977: Sanger sequencing
• 1980’s: Phosphoramidite DNA synthesis
• 1983: PCR to selectively amplify DNA
• 1975: Asilomar – voluntary moratorium on risky applications of
recombinant DNA technology
62
64. Genetic engineering: synthetic biology
New tools à New name
• Parallel DNA sequencing (NGS)
• Long read sequencing (Pacbio,
Oxford Nanopore)
• Short read sequencing (Illumina)
• Today: read 600B bases for $10k
• DNA synthesis
• Primer oligos
• Custom gene synthesis
• Parallel oligo synthesis (oligo pools)
• 250,000 oligos at 230-300nt
• 75M bases for $5-20k
63
65. What challenges face a genetic engineer?
• Proteins are complex
• At present we can’t simulate or model that
complexity with low-level atomic models
• Protein interactions are poorly characterized
• Parameters for mid-level systems biology
models are not accurately measured and many
interactions are unknown
• Many higher levels of complexity
• From cells to tissues to organs to bodies
64
MAPK, cellsignal.com
66. Well then how does
Nature engineer?
65
Influenza, David Goodsell
67. Evolution is an algorithm
• With a goal of quickly finding improved sequences
• In nature: without any information whatsoever about how these
sequences work
66
68. Nature’s algorithm: Darwinian evolution
67
Random
mutation
Natural
selection
Natural rates of mutation
& recombination
Boltzmann
function of fitness
69. Directed evolution uses artificial selection
68
Lab control over
rates of mutation
& recombination
Often defaults to
greedy selection
Random
mutation
Artificial
selection
70. Engineering AAV capsids with directed evolution
69
Wang, Nat. Rev. Drug Disc. 2019
Random
mutation
Artificial selection
(mice, cells, primates)
Random
recombination
71. Why is AAV capsid engineering so challenging?
• As number of mutations
increases, probability of viability
with random search decreases
(exponentially)
• And … AAV needs to be
optimized across multiple
properties
Distance from WT
1 2 3 4 5
Probabilityofviability
Distance from WT
1 2 3 4 5
Probabilityofviability
A&B&C
AB
C
70
72. Random AAV libraries are typically “shallow”
Serotype A Serotype B
100-300 mutations
1-4 changes (~1e18 mutants) 1-4 changes
What’s needed is an efficient algorithmic
exploration of this space
Most useful areas of sequence space are unexplored
71
73. New technologies enable Super-Darwinian algorithms
72
Fitness
Number of library members tested
Super
Darwinian
evolution
Darwinian
evolution
74. Building smarter libraries with direct DNA synthesis
73
Print any DNA sequence
Currently: batch size is ~250,000
sequences up to 300nt
Limitation in length due to low
tolerance for deletions
76. Balancing exploration and exploitation
75
Random
mutation
Artificial
selection
Optimize for exploration
vs exploitation depending
on how many rounds of
selection remain
Sampler
77. Modeling fitness landscapes with machine learning
76
Random
mutation
Artificial
selection
Machine learning models
evaluate a large number
of sequences
Sampler Model
evaluation
81. At Dyno Therapeutics we are building a machine-guided
AAV capsid engineering platform
80
Founded 2018, VC funded, based in Cambridge, MA
82. In contrast to traditional random approaches,
Dyno is systematically searching sequence space
Before Dyno
Random
High throughput
DNA Synthesis
Enrichment via
passaging
High throughput
DNA Sequencing
Single property Multiple properties
Library design
Selection strategy
Optimization
Machine
Learning
81
85. All experiments and data from Harvard (2015-2018)
with George Church and collaborators
84
George
Church
Pierce
Ogden
Sam
Sinai
86. • All possible codon changes
• Insertions
• Substitutions
• Deletions
• Includes barcode replicates and
internal controls
• Testing ~200,000 mutant
genomes in a single animal
• Emphasis on maximizing
information gained in the first
round of experiments
0 mutations
(WT)
1 mutation
Dyno platform: Wide search
85
87. AAV Capsid
GeneMutation Barcode
Wide search: Maximizing information gained
from a single experiment through multiplexing
YY
Y
Y
Y
Y
Capsid
packaging
Immune
evasion
In vivo
delivery
Tissue
specificity
NGS NGS NGS NGS 86
91. Dyno platform: Deep search
0
Distance from WT
1 2 3 4 5
(WT)
90
• Explore far away from natural
capsids by introducing many
mutations in combination
• Emphasis on improving function
based upon information gained
from previous rounds of
screening
92. AAV2
Can we explore deep into sequence space?
Results: still viable at
10-15 mutations
away from WT
=
50-64% similarity
within library region
Deep search pilot
• 80,000 mutants
• 28 amino acid region
• ~10,000 random
• ~15,000 internal replicas
• 1-42 mutations
Deep Search
91
93. Deep Search accelerates AAV engineering
Deep search
library
Random
library
Probabilityofviablepackaging
Distance from WT
92
Deep Search
94. Deep Search accelerates AAV engineering
Deep search
library
Random
library
Probabilityofviablepackaging
Distance from WT
+ deep
learning
+ future
rounds
93
Deep Search
95. Frontiers: Machine-guided capsid engineering
94
Efficient delivery to target cells and tissues
Reduced off-target delivery
Robust evasion of pre-existing immunity
Increased packaging size
High-titer manufacturing capabilities
Natural
capsids
✓
✗
✗
✗
✓
Ideal
✓
✓
✓
✓
✓
All of
these will
be solved
Up to 6kb
for AAV
96. Frontiers in genetic engineering for therapeutics
• Payload engineering
• Cell and tissue specific promoters
• Control over expression dynamics
• Machine-guided engineering
• Exploration strategies
• Machine learning architectures
• Transfer learning from other proteins
• Integration of data from diverse properties
• Technology development
• Advanced methods for DNA assembly and sequencing
95
97. Summary
• Evolution is an algorithm
• Darwinian evolution is random mutation + natural selection
• Directed evolution means artificial selection
• New technologies enable Super-Darwinian evolution
• Dyno Therapeutics is developing a machine-guided capsid engineering
platform
• Now hiring RA’s, Software engineers and experts in AAV gene therapy
• Major challenges in delivery with AAV capsids will be solved soon
• Machine-guided approaches will soon dominate genetic engineering
96
99. Focusing on predictions rather than opinions
• Genome editing and gene therapy give us the ability to cure genetic
diseases and improve health
• Therapies that improve health may also enhance capabilities
• Based on today’s evidence, what is likely to come next?
98
100. Questions to consider
• Will genome editing and gene therapy become mainstream components
of a healthy lifestyle?
• What are the ethical concerns?
• When will these treatments be safe?
• When will benefits outweigh risks?
• When will benefits and risks be distributed justly and equitably?
99
101. Technological revolutions
• Once radical technologies are
now common
• Agriculture, Writing, Metals,
Medicine, Machines, Computers,
Internet
• Once radical behaviors are now
acceptable
• Ethical concerns on risks and
resources often have technical
solutions
• Technical revolutions flourish
under the current model of
economic liberalism
100
102. 1. Germ-line editing with present day tools will not
become mainstream
• The option for embryo selection make germline editing unnecessary
• Germline editing has risks
• Our individualist society will most likely not support experiments that
violate genetic agency
101
103. 2. Somatic therapies will predominate
• Somatic gene therapies are already successful and many more are under
development
• Gene therapy technology will develop faster than human generation time
• There will be no advantage to “locking in” a genetic change using germ-line
genome editing
102
104. 3. Somatic therapies will redefine healthy
• Mono-genetic diseases will be cured through embryo selection, genome
editing and gene therapy
• Treatments will expand to polygenic and non-genetic diseases
• Mainstream therapies will lower costs and reduce risks
• Our individualistic society will tolerate genetic experimentation as a
form of self expression
• DIY somatic gene therapies will pioneer new genetic lifestyles
• The free market will develop mainstream somatic gene therapies
103
105. 4. Genome synthesis will gradually replace ex-vivo
genome editing technologies
Surgery is to small molecule drugs
as
ex vivo cell therapy is to in vivo gene therapy
104
making a small number of changes complete rewriting
Editing Synthesis
With time, editing will introduce more changes, until
eventually we may skip editing in favor of synthesis
106. But applications of whole genome synthesis
technologies will depend on many factors
• Whole genome synthesis obsoletes the function of the germ line
• Disruptive potential of whole genome synthesis is far beyond that of
germ-line genome editing
• Whether genome synthesis will replace sexual recombination is
challenging to predict
• When will society embrace somatic therapies aimed at lifestyle improvement?
• When will whole genome synthesis capabilities be developed?
105
107. 5. Genetics will become self-expression
• Humanity has always been defined more by behaviors than by genetics
• Genome editing, gene therapy and whole-genome synthesis
technologies will make genomics a personal choice
106
109. Advice for scientists
• Move to Cambridge/Boston
• Why?
• Power laws: the best people
want to work with the best
people
• Boston area = Highest
density of talent, capital and
biotech innovation in the
world
108
Especially in gene therapy and genome editing:
Most big Pharma have R&D headquarters,
likewise for CRISPR companies
110. Advice for entrepreneurs
• Major challenges limiting the high potential of these technologies:
• Payment
• Distribution
• Manufacturing
• Delivery
• Redose
• Packaging size
• Payload
• Control over dynamics
• Sensing and actuation
• Polygenic diseases and beyond
109
111. Advice for investors
• Plenty of opportunities will be revealed by learning the science
• ASGCT and ESGCT conferences are a good place to start
• More smaller bets on disruptive technologies (vs incremental
technologies)
110
112. Final conclusions
• Millions of patients have genetic diseases that are untreatable today
• Genome sequencing, genome editing and gene therapy technologies
give us the tools to cure these diseases
• Somatic therapies will predominate over germline editing
• Progress in treating mono-genetic diseases will lead to advanced
treatments for polygenic and non-genetic diseases
• In the long term, individual experimentation, market forces, reduced
cost and reduced risk will make cell therapy and gene therapy part of a
mainstream healthy lifestyle
111