The document discusses advancements in genome editing technologies, highlighting the need for effective therapies for genetic diseases and the challenges associated with delivering these therapies in vivo and ex vivo. It covers various approaches such as viral and non-viral delivery methods, RNA modification therapies, and the application of CRISPR/Cas9 technology for treating conditions like hemophilia and hereditary diseases. Additionally, it emphasizes the importance of precise gene editing and the limitations related to off-target effects and delivery mechanisms.

1. Delivery technologies for genome editing
1

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

10. 10

11. 11

12. 12

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

20. 20

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

23. 23

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

25. Classifications
 Ex vivo or in vivo
 Viral or non-viral
25

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

28. Viral vectors
28

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

32. 32

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

40. In vivo approaches
40

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

46. 46

47. Nonviral
47

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

64. 64

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

69. 69

70. 70