rescue of cftr function in primary bronchial epithelial

Rescue of CFTR function in primary bronchial epithelial cells from patients with cystic fibrosis using lipid nanoparticle delivery of RNA-based therapies

WS09 Workshop 9 -Future therapeutic approaches and insights into our current practice

Michael Torres, PhD ([email protected])VP of R&DJune 11, 2021

NON-CONFIDENTIAL 2

Conflict of interest disclosure I have no, real or perceived, direct or indirect conflicts of interest that relate to this

presentation. I have the following, real or perceived direct or indirect conflicts of interest that relate to

this presentation: Affiliation / financial interest Nature of conflict / commercial company name

Employee and shareholder ReCode Therapeutics, Inc.

This event is accredited for CME credits by EBAP and speakers are required to disclose their potential conflict of interest going back 3 years prior to this presentation. The intent of this disclosure is not to prevent a speaker with a conflict of interest (any significant financial relationship a speaker has with manufacturers or providers of any commercial products or services relevant to the talk) from making a presentation, but rather to provide listeners with information on which they can make their own judgment. It remains for audience members to determine whether the speaker’s interests or relationships may influence the presentation. Drug or device advertisement is strictly forbidden.

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NON-CONFIDENTIAL

• tRNA for nonsense mutations• mRNA (mutation-independent)• Gene editing

FOCUSED ON DELIVERING GENETIC MEDICINES TO PATIENTS

NOVEL LNP PLATFORM SUPPORTS ALL PROGRAMS

3

Primary Ciliary Dyskinesia

• mRNA• Multi-

gene/drug pipeline

CysticFibrosis

Delivery to CF patient-derived hBE* cells

Rescue of CFTR function in hBE cells

Aerosol delivery onto hBEsand into the lungs of mice

Generally well-tolerated in human cells, mice, NHPs

hBE: human bronchial epithelial

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RCT223 (tRNA) for Arginine Opal Nonsense Mutations

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tRNA Payload• Chemically synthesized• Specific for nonsense mutations• Reads premature stop

(nonsense) codon as signal for arginine to make a full-length functional CFTR protein

Formulation• Proprietary lipid nanoparticle

(LNP) optimized for tRNA• Formulation well tolerated in rats

and patient-derived human bronchial epithelial cells

Delivery• Delivered as an aerosol using a

commercially available mesh nebulizer

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Mutation-Independent CFTR mRNA

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mRNA Payload• Optimized sequence for

improved stability, quality and translation efficiency

• Modified nucleotides for reduced immunoreactivity

Formulations• Proprietary lipid nanoparticle

(LNP) optimized for mRNA• Compatible with nebulization• Formulation well tolerated in

mice and patient-derived human bronchial epithelial cells

Delivery• Delivered as an aerosol to the

respiratory epithelium using a commercially available mesh nebulizer

CFTR

RCT223 (tRNA) for Arginine Opal Nonsense Mutations

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0

5

10

15

25 40 54 69 840

5

10

15

11 26 41 56 70 85CFT

R F

unct

ion

Ieq

(µA)

CFT

R F

unct

ion

Ieq

(µA)

0

5

10

15

24 39 53 68 84CFT

R F

unct

ion

Ieq

(µA)

Time (min)Time (min)Time (min)24 hr timepoint 48 hr timepoint 72 hr timepoint

CFTR Function (AUC/cm2/min)

DMSO RCT223

RCT223 (tRNA) Restores CFTR Function in Patient-Derived CF hBE Cells with a Single Administration

Selected traces

Forskolin 10 µMVX-770 1 µM

Benzamil 6 µM

Bumetanide 20 µM

000


Benzamil 6 µM

Bumetanide 20 µM


Benzamil 6 µM

Bumetanide 20 µM

RCT223 RCT223RCT223

DMSO DMSODMSO

Genotype: R553X/F508delTreatment: single apical dose, 12 µg/well tRNAEffect persists for at Least 72hrs

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0

10

5 19 32 45 590

10

5 19 32 45 59

CFTR Function Increases Over Time with 2x/week Dosing of RCT223 (tRNA) in Patient-Derived CF hBE Cells

8

Dose 1 Dose 2

Time (min) Time (min)

CFT

R F

unct

ion

Ieq

(µA)

0

10

5 19 32 45 59

Dose 3

CFT

R F

unct

ion

Ieq

(µA)

CFT

R F

unct

ion

Ieq

(µA)

Time (min)

Benzamil 6 µM


Bumetanide 20 µM

Benzamil 6 µM


Bumetanide 20 µM

Benzamil 6 µM

Forskolin 10 µMVX-770 1 µM Bumetanide 20 µM

RCT223 RCT223

RCT223

DMSODMSO DMSO

• Increasing activity with repeated administrations• Longer term studies are ongoing

Genotype: R553X/F508delTreatment: 12 µg/well tRNA every 3 days (apical)Measure of CFTR function: 24 hrs post treatment

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CFTR Function Increases Over Time with 2x/week Dosing of RCT223 (tRNA) in Patient-Derived CF hBE Cells

9

0

1

2

3

4

DMSO 0.2% RCT223 tRNA 12µg

I eq

µA c

m-2

min

-1

Dose 1Dose 2Dose 3


• Increasing activity with repeated administrations• Longer term studies are ongoing

Genotype: R553X/F508delTreatment: 12 µg/well tRNA every 3 days (apical)

Mutation-Independent CFTR mRNA

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Aerosol Delivery of LNP-formulated mRNA to CF hBEsUsing a VitroCell Exposure System

VitroCell Exposure System and human bronchial epithelial ALI model used for nebulization feasibility/device compatibility, in vitro potency & cell tropism studies

RTX0001Untreated

• Fully-differentiated CF hBE cells• 4 µg/well dose Tomato Red mRNA

• LNP formulated mRNA penetrates CF mucus• High levels of expression distributed throughout the well after a single delivery• VitroCell Exposure system used for delivery of CFTR mRNA to CF hBEs

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LNP Formulated CFTR mRNA Produced CFTR Protein and Restores Function in Patient-Derived CF hBE Cells

12

0

10

20

30

40

50

0 13 27 40 53

24 hour timepoint

RTX0001 CFTR mRNA

DMSO

*#Trikafta

CFT

R F

unct

ion

Ieq

(µA)

Time (min)

Benzamil 6 µM

#VX-809


Bumetanide 20 µM

Immunoblot from primary CF hBEcells

A single, low dose significantly restores CFTR function

Band CBand B

Actin

*VX-661 3.3 µM + VX-445 3 µM. #Added to BL media

Genotype: G542X/F508delTreatment: single administration of 4 µg/well CFTR mRNA delivered as an aerosol

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0

10

20

5 19 32 45 590

10

20

5 19 32 45 59

13

Dose 1 Dose 2

Time (min) Time (min)

CFT

R F

unct

ion

Ieq

(µA)

0

10

20

5 19 32 45 59

Dose 3

CFT

R F

unct

ion

Ieq

(µA)

CFT

R F

unct

ion

Ieq

(µA)

Time (min)

Benzamil 6 µM


Bumetanide 20 µM

Benzamil 6 µM


Benzamil 6 µM


RTX0001 CFTR mRNA

RTX0001 CFTR mRNA

RTX0001 CFTR mRNA

DMSO DMSO DMSO

Rescued CFTR Function in CF-Derived hBEs is Maintained with 2x/week Dosing of LNP-formulated CFTR mRNA

CFTR activity is maintained with repeated administrations

Genotype: G542X/F508delTreatment: single administration of 4 µg/well CFTR mRNA delivered as an aerosol

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The RTX0001 mRNA Response is Durable in Patient-Derived CF hBE Cells and Supports 2x/week Dosing

14RTX0001 delivered as an aerosol. Dosing frequency every 3 days.


Rescue is maintained with repeated administrations

0

1

2

3

4

5

6

7

8

DMSO 0.2% RTX0001 CFTR mRNA 4µg

I eq

µA c

m-2

min

-1

Dose1Dose 2Dose 3

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Ciliated Cells Basal Cells Club Cells Goblet Cells0

20

40

60

80

100%

TR p

ositi

ve c

ells

Cell Subsets in HBE cultures

SORT000014A3-SC7 20% DODAP, 40:1

ReCode LNP RTX0001 Delivers mRNA to the Target Cells in Primary hBECells and Lung Tissue in Mice

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Cell type Marker

Club Secretoglobin Family 1A Member 1 (SCGB1A1)

Goblet Mucin 5AC (MUC5AC)

Basal Cytokeratin 5 (CK5)

Ciliated Acetylated-Tubulin (AC-Tubulin)

Aerosol Delivery of Luciferase mRNA and Protein Expression in the Lungs of Mice

Doses represent the amount of Luc2 mRNA delivered into the exposure chamber by nebulization. Estimated (not measured) per mouse delivered dose is 0.01, 0.06 or 0.22 mg/kg

1Study of SORT-LNP delivered as an aerosol encoding Tomato Red Protein in a primary ALI hBE model.

Tomato Red Protein Expressed in Target Cell Types in hBE Cells1

RTX0001

0.4 mg RNA

2.0 mg RNA

8.0 mg RNA

• LNP formulation compatible with Aerogen solo mesh nebulizer

• Good distribution and expression in mouse lungs after a single administration

• Treatments were well tolerated in all mice• Using the same LNP formulation, protein expression

has been detected in the target cells of NHP lungs with a single administration of nebulized mRNA

NON-CONFIDENTIAL

Conclusions

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mRNA Replacement Approach

Functional restoration achieved in the CF patient-derived hBE model

Responses are durable mRNA delivered to CF target cells and

protein produced Formulations well toleratedNext Steps: NHP studies in 2021

tRNA Approach for Nonsense Mutations

Functional restoration achieved in the hBE model Both as a single administration and

with repeat dosing Formulations well tolerated in CF

patient-derived hBE cultures

Next: Rodent studies in 2021

NON-CONFIDENTIAL

• ReCode Therapeutics⎼ Dmitri Boudko⎼ Ella Meleshkevitch⎼ Melissa Coquelin⎼ Jack Eby⎼ Xueliang Yu⎼ Paul Gao⎼ Daniella Ishimaru⎼ Mirko Hennig⎼ Rumpa Bhattacharjee⎼ Vladimir Kharinotov⎼ Brandon Wustman⎼ David Lockhart

Acknowledgements

• CF Foundation⎼ Martin Mense⎼ William Skach

• Advisors⎼ Philip Thomas⎼ Robert Bridges⎼ Daniel Siegwart

• Funded in part through a grant by the CF Foundation

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rescue of cftr function in primary bronchial epithelial

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