2. Effective therapies for many genetic diseases
are still needed
>3,000 human genes associated with mendelian diseases .
~4,000–7,000 additional disease-associated genes will be
uncovered in the next decade.
<5% of rare diseases have an effective treatment
It is difficult to use small-molecule, BMT or surgical approaches
to treat most genetic diseases
2
3. Protein therapeutics for treating certain
genetic disorders
1. Protein replacement/augmentation therapies
2. Antibodies
To treat disorders for which a deficient protein functions at least
partially in the extracellular milieu
3
4. Most of these pr therapeutics target a limited
number of biomolecules that are involved in a
small proportion of genetic diseases
Therapeutic protein Condition
Recombinant acid α-glucosidase Pompe
Recombinant factors VIII Haemophilia A
Recombinant factors IX Haemophilia B
Humanized vascular endothelial growth
factor A (VEGFA)-specific antibody
Age-related macular degeneration
4
5. AAV-mediated delivery of a functional gene
tiparvovec (AAV1-LPLS447X) for LPLD
The first gene therapy to receive marketing authorization in
Europe
In 2012 became the first gene therapy product based on viral
gene-transfer technology received marketing approval in Europe.
$1 million per treatment
5
6. RNA modification therapies: RNAi & ASOs
Product Condition Status
ASO-based
product
Homozygous familial
hypercholesterolaemia
FDA approved
Alicaforsen Pouchitis Advanced stages
Other RNA
modification
therapies
Transthyretin-mediated
amyloidosis
Phase III
6
7. Limitations of ASO & RNAi and AAV gene
transfer
Delivery barriers in a subset of organs and tissue types
Incomplete suppression of disease proteins
Off-target effects
Safety concerns (gene transfer):
Genomic integration
Mutagenesis
Oncogenesis
7
8. AAVs facilitate gene transfer and episomal
expression in non-dividing cells, but:
Expression decrease over time (cell turnover)
Loss of episomal genomes in growing tissues of paediatric patients
Lack of precise control of therapeutic genes
Unusable for gain-of-function genetic diseases
Limited size of encapsulated transgenes
Pre-existing immunity
CD8+ T cell-mediated adaptive response against the AAV capsid
8
9. Genome editing in contrast to gene-transfer
approaches, uses programmable DNA nucleases
Precisely modification of genome
Potential to treat and even cure a range of important diseases via:
Deletion of disease-prone sequences
Correction of mutations
Site-specific insertion of therapeutic genes
9
13. Specificity & potential for off-target effects
Off-target can lead to
Unwanted mutations
Oncogenes
Tumor suppressors
DNA repair genes
Potential toxicity
Specificity may also be affected by
Dosage
Expression pattern of tools
Number, type and stage of edited cells 13
14. Genome editing for genetic diseases
Treatment of monogenic diseases vs multigenic diseases
Each monogenic disease may require various efficiencies of
editing:
Restoration of 3–7% F9 to reverse symptoms of haemophilia B
α1-antitrypsin deficiency may require >30% gene correction in the liver
14
15. Factors affect on treatment
Indel formation vs HDR repair
The nature of the cellular target:
HSC vs mature cells
Effective delivery
Immune response to the corrected genes
~30% of patients with haemophilia A who received protein therapy
developed inhibitory antibodies
15
16. All the tools can create DSBs
Two cellular endogenous DNA repair pathways:
HDR (with template (donor) DNA)
NHEJ
16
17. NHEJ can disrupt disease-causing genes or restore
the reading frame of a dysfunctional gene
Efficient but error-prone
Small “indels” at the desired genomic locus
Shifting the reading frame
Production of nonfunctional proteins
17
18. Many diseases requiring gene correction or
addition (by HDR) rather than gene inactivation
Nature of the cell (HDR is largely restricted to cells at S/G2 phases )
Optimization of the conditions of delivery may facilitate enhanced HDR
Timing of providing the donor template
Format and dose of the donor
Synchronization of the cell cycle
Suppressing NHEJ
18
19. The average mutation rate of TALEN and CRISPR
is substantially higher than that of meganucleases
and ZFNs
Meganucleases and ZFNs: low efficiencies in inducing site-
specific DNA breaks
TALENs and CRISPR: ~80–90% success rates in mammalian
cells to induce >1% mutation frequencies
19
21. Multiplex gene editing
ZFNs & TALENs:
Mismatched dimers can form off-target
CRISPR
High efficiency
Without substantially increasing the required dose
21
22. The most substantial challenge is the safe and efficient
delivery of genome-editing biomacromolecules
Delivery of genome editing vs ASO, RNAi & “classic gene transfer”
One or a limited number of doses could be sufficient
Classical gene replacement therapy: long-term transgene expression
RNAi therapy: repeated dosing
Multiple components must be delivered to the nucleus
22
24. For HDR-mediated gene editing, it is also necessary to provide a donor ssDNA or
dsDNA template
Nucleases CRISPR–Cas system
In the format of DNA, mRNA or
protein
• Meganucleases: ~1 kb
• ZFNs: two molecules ~1 kb each
• TALENs: two molecules ~3 kb each
Cas in the format of DNA, mRNA
or protein
Guide RNA, in the format of DNA
or RNA
• ~3.5–4.5 kb
Genome editing tools: size & components
24
26. Genome-editing systems can be delivered ex
vivo or in vivo
Ex vivo
Some nanomaterial and integrating
lentiviral vectors, may not be
suitable (low efficiency or safety
concerns)
Use of physical methods that are
not suitable for systemic
application:
In vivo : Additional hurdles
Viral:
Broad tropism
Long-term transgene expression
Lipid-based and polymer-based
nanoparticles and conjugate technologies
for RNA
26
27. Delivery of biomacromolecules: viral or non-viral
Viral vector to encapsulate a
gene, in RNA or DNA form
Lentiviruses
Adenoviruses
AAVs
Non-viral delivery
Physical methods
Electroporation
Microfluidic-based technologies
Nanomaterial-based methods
Cationic lipids
Cell-penetrating peptides (CPPs)
Self-assembled nanoparticles
27
29. Viral vectors with ssDNA may generate higher HDR rates than
dsDNA or plasmid DNA
29
30. As opposed to lentiviruses, adenoviral DNA does not integrate
into the genome and is not replicated during cell division
IDLVs
Low risk of insertional mutagenesis
Delivery to various human cell types
Free-ended, ds genome: insertion at off-target sites or spontaneous DSBs
Adeno:
Several infections
Neutralizing antibodies
Adenoviruses that infect different species
30
31. AAVs: random integration with a negligible
frequency
Stable integration at a specific site (AAVS1in chromosome 19)
Gene is inserted between the ITRs that aid in concatemer
Episomal nonreplicative concatemers
31
33. IDLVs have been used to deliver both ZFNs
and donor templates in vitro
Mostly episomal
Gradually diminishes by dilution in dividing cell
Short-lived expression of ZFNs
In HEK293T and Epstein–Barr virus-transformed B lymphocytes: High
efficiency
In CD34+ HSPCs:
Without hotspot: Low (~0.1% of total cells)
Viral ---- In vitro and ex vivo
33
34. Adenoviral and AAV vectors
Adenoviral vector more efficiently expressed ZFNs in primary T cells than an IDLV
Delivering ZFNs in HSCs from most to least efficacious:
mRNA electroporation > plasmid electroporation > adenoviral vector > IDLV
20–40% gene addition in HSPCs by:
Electroporation of ZFN mRNA
AAV6-mediated delivery of the donor template
Viral ---- In vitro and ex vivo
34
35. 8–60% rates of HDR in primary human T cells by:
mRNA of megaTAL
Donor in AAV
Viral ---- In vitro and ex vivo
35
36. Applications of ex vivo gene editing
Gene-edited HSCs could be used to treat a range of genetic
blood disorders
Modified primary T cells are promising tools for cancer
immunotherapies
Insertion of CARs
Deletion of PD1
Universal T cells
Ex vivo applications
36
37. Genome-editing tools for inserting CARs
Integrating viral vectors to insert CARs into the genome
Semi-random integration
Safety concerns
Alternative: genome-editing tools to insert CARs specifically at a
designated site
Ex vivo applications
37
38. Genome-editing tools to make universal T cells
Personalized process is expensive
Difficulty of generating high numbers of healthy T cells
Reducing immune rejection by knocking out genes involved in
immune surveillance (eg. HLA).
Increased consistency
Increased availability
Increased potency
Increased safety
Ex vivo applications
38
39. Effective levels of HDR in human primary T cells by:
Electroporation of ZFN mRNA
AAVs delivery of donor template
39
41. AAV delivery of ZFN for treatment of hemophilia
Small size of ZFNs facilitates their packing into AAVs
AAV-8 delivery of ZFNs and donor
Gene targeting in the liver
To correct factor IX.
Integration of gene into Albumin locus
Long-term expression of FVIII and FIX
Useful for the treatment of lysosomal enzyme deficiencies
Viral ---- In vivo, ZFN
41
42. Dual-vector system for in vivo targeting of
single & multiple genes in mouse brain
SpCas9 and sgRNA expression cassettes into two
separate AAVs
Packaging of SpCas9 with:
Minimal polyadenylation signal
Truncated version of the neuron-specific promoter
Viral ---- In vivo CRISPR
42
43. A single AAV-8 vector for packaging of sgRNA &
SaCas9
Thyroxine-binding globulin promoter/SaCas9
sgRNA (targeting PCSK9) expression cassette
>40% indel formation at the Pcsk9 locus in liver
Significant decrease in serum PCSK9 and total cholesterol
Viral ---- In vivo CRISPR
43
44. Adenovirus-mediated delivery or dual AAV vectors
for treatment of DMD model
The mutated exon 23 of DMD was deleted
Partially restoring DMD expression in skeletal muscle
Enhancing skeletal muscle function
Viral ---- In vivo CRISPR
44
45. Reversing the mutations of the ornithine
transcarbamylase in newborn mice
Two AAVs
One expressing SaCas9
The other carrying a sgRNA & donor
Viral ---- In vivo CRISPR
45
48. Non-viral methods advantages
Can avoide the transcription process
Low immunogenicity
Precise control of the duration
Transient expression
Decrease the risk of off-target effects & tumorigenesis
Nucleotide chemical modifications,
Viral nanoparticles engineered based on native viral structure
Repeated administration
Improved efficacy
Potential to transfer large genetic payloads
48
49. The most widely investigated physical transfection
method is electroporation
Electroporation of Cas9 and two different sgRNA-encoding plasmids
significant gene deletion in HSPCs and in primary T cells
Electroporation of [Cas9 + in vitro transcribed sgRNA ] RNP
Higher efficiency genome editing
Decreased off-target effects
Less cellular toxicity
Non-Viral ---- In vitro & ex vivo
49
50. Electroporation of Cas9 mRNA and sgRNA
resulted in modest gene editing
)instability of unmodified sgRNA(
Non-Viral ---- In vitro & ex vivo
50
51. Mechanical deformation method by constricting
channels: ‘squeeze’ delivery
Microfluidic devices with dimension smaller than the diameter of
specific cells
Diffusion of target materials to through transient membrane pores
Non-Viral ---- In vitro & ex vivo
51
52. Mechanical deformation of cells for
CRISPR-mediated efficient gene editing
ssDNA, siRNAs, and large-sized plasmids into different cell types:
Adherent and non-adherent cells (HEK293T, MCF7, SUM159, SU-DHL-1)
Hard-to-transfect lymphoma
Embryonic stem cells
“Squeeze” delivery of RNPs to HSPCs:
Significantly higher than electroporation
Less toxicity
Non-Viral ---- In vitro & ex vivo
52
53. iTOP for Direct intracellular pr delivery:
hypertonicity-induced micropinocytosis+ propanebetaine
iTOP-mediated delivery of
recombinant Cas9 pr & sgRNA
High levels of indel formation in cells
Non-Viral ---- In vitro & ex vivo
53
54. Nanoparticle-mediated delivery: synthetic
lipid-based or polymer-based delivery vectors
The efficiency is often dependent on the type and status of the cell
Commercially available transfection reagents for use in cell culture
Cationic liposomes to deliver Cas9 and sgRNA-coding plasmids
Cationic lipids + RNPs or engineered negatively charged TALE: higher
gene-editing efficiency than with nucleic acid complexes
Non-Viral ---- In vitro & ex vivo
54
55. Conjugating Cas9 proteins and sgRNAs to CPPs
may facilitate intracellular delivery
• Embryonic stem cells
• Dermal fibroblasts
• HEK293T cells
• HeLa cells
• Embryonic carcinoma cells
• Efficient gene disruptions
• Reduced off-target mutations
relative to plasmid transfections 55
56. Conjugation of R9 to a surface-exposed Cys
on TAL effector repeat
Reversible under reducing
conditions
Knockout of the human
CCR5 and BMPR1A genes
at rates comparable to
those achieved with
transient transfection of
TALEN expression
vectors
56
57. Delivery of ZFNs without any delivery vectors was
reported to disrupt endogenous genes
In several cell lines and in primary CD4+ T cells
Intrinsic cell-penetrating capabilities
Effective concentration is at micromolar levels
Non-Viral ---- In vitro & ex vivo
57
58. Hydrodynamic injection
the first in vivo use of CRISPR: for treatment of
tyrosinaemia
Transient tissue damage
Limited use in clinical trials
Approximately 0.4% of hepatocytes were corrected
Sufficient to rescue
Because the repaired hepatocytes have a growth advantage
Non-Viral ---- In vivo
58
59. Physical methods
Electroporation of Cas9 & sgRNA plasmid into skeletal muscles
Restored function of local tissue in a mouse model DMD
Non-Viral ---- In vivo
59
60. Lipid nanoparticle-mediated delivery of Cas9 mRNA
+ AAV encoding an sgRNA & donor template
HDR-mediated correction occurred in >6% of liver cells
Off-target nuclease activity was below the limit of detection using
an unbiased genome-wide analysis
Non-Viral ---- In vivo
60
61. Nanoparticles
Intratracheally delivered chitosan-coated poly(lactic-co-glycolic)
acid (PLGA) nanoparticles encapsulating ZFN mRNA
AAV encoding a constitutive CAG promoter to target the site
where the SP-B cDNA was under the control of an inducible
promoter
Non-Viral ---- In vivo
61
62. Cas9–sgRNA complex was loaded into DNA-based self-
assembled NPs (DNA nanoclews) coated with PEI
Non-Viral ---- In vivo
62
63. Fusing therapeutic pr to negatively charged pr &
complexes with cationic transfection reagents
Cre or TALE transcription activators tagged with GFP
Nanomolar concentrations
Complexation with lipofectamine
Cas9–sgRNA and lipofectamine complexes
Genetic modifications of inner ear cells in vivo
Non-Viral ---- In vivo
63
65. Injection of genome-editing systems into
animal embryos and zygotes
Co-injection of DNA donors with Cas9 mRNA and sgRNAs
65
66. Current status
HIV gene therapy:
CCR5 modification of CD4+ T cells & HSPCs by ZFN
Gene therapy for haemophilia B
AAV delivery of ZFN & donor template of FIX
Gene therapy for mucopolysaccharidosis I&II
AAV delivery of ZFN & donor template of IDUA & IDS
66
67. Technologies Delivery Disease
Treatment
type
Company
CRISPR–Cas9
Lipid
nanoparticles
Liver diseases: transthyretin amyloidosis, α1-
antitrypsin deficiency, hepatitis B virus and
inborn errors of metabolism
In vivo
Intellia
Therapeutics
HSCs and CAR T
cell programmes by
CRISPR–Cas9
Electroporation - Ex vivo
Intellia
Therapeutics
CRISPR–Cas9 &
CAR T cell
AAV-mediated
local delivery
Eye disease In vivo
Editas
Medicine
CRISPR–Cas9
CRISPR–Cas9 &
CAR T cell
-
Haemoglobinopathies,
Immunodeficiencies, B-cell malignancies
ex vivo
CRISPR
Therapeutics
CRISPR–Cas9 -
diseases in the
liver, eye, lung and other organs
In vivo
CRISPR
Therapeutics
67
68. CRISPR Therapeutics
http://www.crisprtx.com/programs/pipeline
Our lead program targeting the blood diseases β-thalassemia and
sickle cell disease has entered clinical testing, as has our first
allogeneic CAR-T program targeting B-cell malignancies. We are
also advancing additional blood stem cell, immuno-oncology,
regenerative medicine and in vivo programs towards the clinic.
68
Adenoviruses have a capacity of ~8.5 kilobases, high levels of protein expression, and transient gene expression. The onset of expression can occur as early as 16-24 hours after infection. The high immune response from the target cells are the main limitation of adenoviral systems. Despite this, they are still widely used in research, due to their highly efficient transduction of most tissue.
AAVs have a packaging capacity of ~4.5 kilobases, relatively low levels of protein expression, and the potential for long lasting gene expression. The tropism of AAV can also be increased via different serotypes. The primary disadvantage of AAV is its smaller packaging size for gene of interest, as well as a much later onset of expression (2-7 days for in vitro and 3-21 days for in vivo). However, this delivery system triggers very low levels of immune response.
These changes, as well as the toxic nature of the pentons, destroy the endosome, resulting in the movement of the virion into the cytoplasm. With the help of cellular microtubules, the virus is transported to the nuclear pore complex, whereby the adenovirus particle disassembles. Viral DNA is subsequently released, which can enter the nucleus via the nuclear pore
Gene therapy with genetically modified human CD34+hematopoietic stem and progenitor cells (HSPCs) may be safer using targeted integration (TI) of transgenes into a genomic ‘safe harbor’ site rather than random viral integration. We demonstrate that temporally optimized delivery of zinc finger nuclease mRNA via electroporation and adeno-associated virus (AAV) 6 delivery of donor constructs in human HSPCs approaches clinically relevant levels of TI into the AAVS1 safe harbor locus. Up to 58% Venus+ HSPCs with 6–16% human cell marking were observed following engraftment into mice. In HSPCs from patients with X-linked chronic granulomatous disease (X-CGD), caused by mutations in the gp91phox subunit of the NADPH oxidase, TI of a gp91phox transgene into AAVS1 resulted in ~15% gp91phox expression and increased NADPH oxidase activity in ex vivo–derived neutrophils. In mice transplanted with corrected HSPCs, 4–11% of human cells in the bone marrow expressed gp91phox. This method for TI into AAVS1 may be broadly applicable to correction of other monogenic diseases
proprotein convertase subtilisin/kexin type 9
Cas9 from Staphylococcus aureus (SaCas9) is ~1 kb shorter than SpCas9
As shown, the small-molecule NDSB-201 is essential for the introduction of native protein into cells. NDSB-201 is part of a group of zwitterionic compounds used to reduce protein aggregation and facilitate