Lentiviral Design for Delivery of Homing Endonucleases

mCherry

HA-NLS-HERSV R U5 hPGK R U5sinU3Repair Template CpptRRE

RRL Lentiviral Backbone

0 102 103 104 105

Texas Red PE-A

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20

40

60

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f Ma

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0 102 103 104 105

FITC-A

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102

103

104

105

Texa

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PE

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0.47 0

1.4298.1

0 102 103 104 105

FITC-A

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102

103

104

105

Texa

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PE

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89.1 3.29

0.0837.54

0 102 103 104 105

FITC-A

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SS

C-A

0.052

0 102 103 104 105

FITC-A

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C-A

96.4

0 102 103 104 105

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C-A

59.8

0 102 103 104 105

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C-A

33.1

0 102 103 104 105

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C-A

11.8

0 102 103 104 105

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C-A

5.96

0 102 103 104 105

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C-A

0.1

0 102 103 104 105

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C-A

62

0 102 103 104 105

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C-A

10.9

0 102 103 104 105

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C-A

5.17

0 102 103 104 105

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C-A

0.86

0 102 103 104 105

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C-A

0.94

0 102 103 104 105

FITC-A

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C-A

0.16

0 102 103 104 105

FITC-A

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C-A

0.29

0 102 103 104 105

FITC-A

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C-A

0.066

0 102 103 104 105

FITC-A

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C-A

0.074

0 102 103 104 105

FITC-A

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SS

C-A

0.051

0 102 103 104 105

FITC-A

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C-A

0.039

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SS

C-A

0.051

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FITC-A

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SS

C-A

94.4

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SS

C-A

50.4

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SS

C-A

29.1

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SS

C-A

10.2

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SS

C-A

5.13

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C-A

0.039

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C-A

20.1

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C-A

2.19

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C-A

0.92

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C-A

0.17

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C-A

0.21

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C-A

0.1

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C-A

0.24

0 10 2 10 3 10 4 10 5

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C-A

0.08

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C-A

0.053

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C-A

0.039

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C-A

0.055

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C-A

0.14

0 10 2 10 3 10 4 10 5

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C-A

93.5

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C-A

48.8

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C-A

27.1

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C-A

8.98

0 10 2 10 3 10 4 10 5

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C-A

4.82

0 10 2 10 3 10 4 10 5

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C-A

0

0 10 2 10 3 10 4 10 5

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C-A

3.14

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SS

C-A

0.46

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SS

C-A

0.16

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SS

C-A

0.042

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SS

C-A

0.06

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SS

C-A

0.028

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SS

C-A

0.058

0 10 2 10 3 10 4 10 5

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C-A

0.11

0 10 2 10 3 10 4 10 5

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C-A

0.042

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C-A

0.028

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C-A

0.015

Integrase Deficient Lenti Virus

0 10 2 10 3 10 4 10 5

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C-A

0.11

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C-A

96.8

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C-A

64.4

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C-A

40.2

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C-A

16.6

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C-A

10.3

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C-A

0.15

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C-A

93.6

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C-A

48.5

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SS

C-A

27.5

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C-A

5.55

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C-A

6.55

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C-A

2.04

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C-A

14.7

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C-A

1.76

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C-A

1.5

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C-A

1.24

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C-A

3.04

psP

AX

2D

64V

untreated 1:10 1:50 1:100 1:500 1:1000

DAY 2DAY 12DAY 8DAY 5

Day 19 post transduction:0.62% of cells remain GFP+

Directions

• Effect of local chromatin structure on the efficiency of cutting and repair

• Role of repair template topology on conversion frequency

HEs for Targeted Integration

A G A T A G G T C A G G G A T A A C A G G G T A A C G C G T A A C A G T

A G A T A G G T C A G G G A T A A C A G G G T A A C G C G T A A C A G T207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242

1696

0

Fragmentmc9_B03

Can an HE induced double stranded break facilitate targeted transgene integration?

Genomic -7C+11C Sce Site in Canine

WT -7C +11C-7C

+11CAni WT -7C +11C

-7C+11C

Ani WT -7C +11C-7C

+11CAni WT -7C +11C

-7C+11C

AniSubstrate:

recSce cleaves +11C miscognante

linearized 5units 0.5units 0.2units

Directions

• Characterize -7C-Sce cleavage of the target site using DR-reporter

• Assess cleavage of the genomic site in-vivo

• Attempt to target reporter to the locus

• 2 nucleotide changes switch fluorophores

Exogenous Template HDR Blue-Green Reporter System

• Swappable HE cleavage site 70bp from target, does not affect reporter • Intron allows use of >1Kb repair template

T Y G

Fluorophore “Switch” Repair Template

Asc1 Age1

eGFP53-238

T Y G eGFP Repair

Product

S H G

Asc1

Age1

eBFP1-52 eBFP53-238Intron

70 bp

eBFP HE Substrate

Swappable HE substrate