POSTER #P.59

mRNA-based Engineering of Red Cell Therapeutics (RCTs) to Generate Potent, Allogeneic Cellular TherapiesUrjeet Khanwalkar, Sneha Hariharan, Clair Depew, Sivan Elloul, Zachary Koncki, Farris Nabulsi, Eric Finnemore, Arjun Bollampalli, Torben Straight Nissen, Omid F. Harandi

Rubius Therapeutics, Cambridge, MA, USA

DNA-based gene therapies that require genome integration are limited by the size of the sequence they encode, number of proteins they can express, and the potential for uncontrolled proliferation. A recent wave of RNA-based gene therapies entering clinical trials has brought us closer to using mRNA as a potential therapeutic nucleic acid. Here, we report how mRNA can be used to engineer red blood cells with long-lasting protein expression to create an entirely new class of cellular medicines called Red Cell TherapeuticsTM (RCTs).

• RCTs are a new class of allogeneic, off-the-shelf cellular therapies for the potential treatment of rare diseases, cancer, and autoimmune diseases (Figure 1)

• RCTs are engineered to express hundreds of thousands of copies of intracellular or membrane-bound therapeutic proteins, such as intracellular enzymes, co-stimulatory molecules and cytokines

• Until now, the RED PLATFORM® has enabled the genetic engineering of donor-derived CD34+ hematopoietic stems cells (HSCs) using lentiviral-based DNA transduction. Here, we report on expanding RCT engineering methods via mRNA-based gene delivery

INTRODUCTION

OBJECTIVES

Figure 1. The RED PLATFORM® Generates Allogeneic, Off-the-Shelf Cellular Therapies

Figure 2. Sustained Protein Expression for 9 Days Post mRNA Electroporation

• Delivered at a dose of <1% of total red blood cell volume in the body

• Universal, scalable, and consistent manufacturing process

To demonstrate:

• Expression of intracellular and/or membrane-bound therapeutic proteins using mRNA-based gene delivery • The expression of a single protein or combinations of multiple proteins using mRNA-based gene delivery• Tunable expression of therapeutic proteins• A scalable process

Figure 2: CD34+ HSCs were cultured using the Rubius RED PLATFORM® to generate RCT progenitor cells, which were then electroporated with eGFP mRNA. Protein expression was monitored by flow cytometry. A: mRNA-based gene delivery results in efficient gene expression (over 97%) in RCTs and is sustained for 9 days. B: Median fluorescent intensity (MFI) was used to measure the amount of protein per RCT.

Figure 3: mRNA-based gene delivery can be used to efficiently express multiple proteins in RCTs. No mRNA control, eGFP mRNA alone, mCherry mRNA alone, or eGFP and mCherry mRNA were electroporated into RCT precursor cells, and the resultant protein expression was observed via flow cytometry and fluorescent microscopy. The fluorescent microscopy was conducted at a 20x magnification.

Figure 4: mRNA-based gene delivery can be used to efficiently express multiple proteins in RCTs. No mRNA control, Rituximab mRNA alone, TRAIL mRNA alone, or membrane-bound Rituximab scFv(Rtx-scFv) and TRAIL mRNA were electroporated into RCT precursor cells, and the resultant protein expression was observed via flow cytometry. Rtx-scFv and TRAIL were stained by fluorescent antibodies and their expression measured by flow cytometry as above.

Figure 6: mRNA encoding for a phenylalanine ammonia lyase-green fluorescent fusion protein (PAL-GFP) was electroporated into RCT precursors in a small scale (1x) and large scale (2000x) reaction and cultured in bioreactors. Protein expression was monitored by flow cytometry.

All authors: Employment with and equity ownership in Rubius Therapeutics.

Figure 5: A: Varying amounts of 4-1BB ligand mRNA were electroporated into RCT precursor cells, which resulted in a controlled titrated amount of 4-1BB ligand expression on each RCT. B: These RCTs were then co-incubated with a 4-1BB-HEK-NF-kB-reporter cell line to measure 4-1BBL-dependent activity via a luciferase assay. A dose-dependent increase in 4-1BB ligand activity was observed, which is controlled by tuning the amount of mRNA.

Figure 3. Efficient Co-expression of Multiple Intracellular Therapeutic Proteins in RCTs

Figure 4. Efficient Co-expression of Multiple Extracellular Therapeutic Proteins in RCTs, Membrane Tethered Rituximab scFv and TRAIL

Figure 5. Tunable Control of Therapeutic Protein Expression in Each RCT

Figure 6. Efficient and Scalable Process for Generating mRNA-based Therapeutic RCTs

Figure 7.mRNA-Based Gene Delivery Can Be Used to Generate a New Class of Cellular Therapy With Sustained Protein Expression

eGFP

mC

he

rry

TRAIL

Rtx

-scF

v

0.01%

97.5%

0% 93.7%

2.71%

0.01%

3.53%2.47%

0%

1.46%

0%

1.27%

87.5%

0%

4.62%

0.3%

84%

79%

2.1%

5.4%

CONCLUSIONS

ACKNOWLEDGEMENTS

DISCLOSURES

• The results demonstrate that mRNA-based gene delivery using optimized electroporation can be used to generate an entirely new class of cellular therapy with sustained protein expression (Figure 7)

• Using mRNA-based gene delivery in RCTs demonstrated efficient co-expression of multiple membrane-bound or intracellular proteins

• mRNA-based gene delivery enables tunable protein expression in RCTs

• mRNA-based engineering of RCTs is scalable

Poster design support was provided by Dennig Marketing Group, sponsored by Rubius Therapeutics.

A special thank you to Tom Wickham, Ph.D., and Lori Melançon of Rubius Therapeutics for input, review and design of the poster.

GENETIC �ENGINEERING �

EXPANSION & �DIFFERENTIATION

RED PLATFORM®

CD34+ HEMATOPOIETIC PRECURSOR CELLS

SINGLE �HEALTHY�O- DONOR

ENUCLEATION & MATURATION

RED CELL THERAPEUTIC

100-1000’s �OF DOSES

mRNA

DNA

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

0 1 2 4 8 16 24 32 40

# 4

-1B

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co

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RC

T

mRNA Amount (mg)

mRNA-based dose-dependent protein expressionA B

1.1

40.0

57.4 60.2

76.183.2

89.6 89.7 91.4

0

10

20

30

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0 1 2 4 8 16 24 32 40

Fo

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mRNA Amount (mg)

Dose-dependent 41BB-L activity

ENUCLEATION & MATURATION

RED CELL THERAPEUTIC

GENETIC �ENGINEERING �

EXPANSION & �DIFFERENTIATION

EARLY�PROGENITOR �CELLS

mRNA

Tunable expression of therapeutic proteins

Multiple membrane-bound therapeutic protein expression

Multiple intracellular therapeutic protein expression

eGFP mRNA

Electroporation

RCT precursors

Analyze for 9 days

RCTs

A

B

0

20

40

60

80

100

0 50 100 150 200 250

% R

CTs

pos

itive

for e

GFP

sig

nal

Hours post mRNA Electroporation

eGFP_RCTs Control

0

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25000

30000

35000

0 50 100 150 200 250

MF

i fo

r e

GF

P (F

ITC

)

Hours post mRNA Electroporation

mRNA2000-Fold

Scaleup

RCTs

Cells grown in 1L bioreactors

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

0 50 100 150 200 250

MF

I fo

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(FIT

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Hours post mRNA Electroporation

0

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0 50 100 150 200 250

%C

ells

po

siti

ve f

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PA

L-G

FP

Hours post mRNA Electroporation

Control Small Scale Large Scale

Electroporation

0%

0.03%

0.01%

99.9% 0.06%5.12%

0.07%94.7%