Eduardo Marbán, MD, PhD gives a deep dive into the complexities of regenerative cardiovascular medicine and the future directions for cell therapies. Dr. Marbán’s lab has found several noncoding RNA (ncRNA) species, including short Y RNAs, which themselves have intriguing biological actions. The ncRNA within extracellular vesicles can either be used as they occur naturally or serve as bioinspiration for new chemical entities. The next generations of cell-free biologics (extracellular vesicles and noncoding RNAs) may provide, or even transcend, the benefits of cell therapy without the intrinsic limitations. Key Topics Include: - Understand the current status of cell therapy for heart disease - Review the mechanisms whereby cells exert their therapeutic benefits - Explore the future of RNA drugs inspired by the contents of extracellular vesicles

1. Copyright 2022. All Rights Reserved. Contact Presenter for Permission
Cardiovascular Regenerative
Medicine: Deconstructing
Regenerative Therapeutics
Eduardo Marbán, MD, PhD
Executive Director
Smidt Heart Institute
Cedars-Sinai Medical Center

2. Partners and Sponsors

3. Cardiovascular regenerative medicine:
deconstructing regenerative therapeutics
Eduardo Marbán, MD, PhD
Executive Director, Smidt Heart Institute
Cedars-Sinai Medical Center
Disclosure:
Founder’s equity in Capricor Inc.

4. Our initial goal with cell therapy (2004)
To repair the “permanently” injured heart
Our updated goals after 18 years of
discovery work (2022)
To use cells, exosomes or novel RNA drugs
as therapeutic candidates for a broad range
of inflammatory/fibrotic diseases

5. Today’s talk: outline
• Discovery of CDCs
• Novel RNA drugs
Predictable,
iterative
steps
Fantasy version How it really happened
Discovery of CDCs
Novel RNA drugs

6. Cell Type Human heart progenitor cell
Characteristics CD105+, CD45-; could differentiate into multiple cardiac lineages
Clinical Trials CADUCEUS-completed-autologous phase 1. Twenty-five patient study
showed regeneration in CDC-treated post-MI subjects with mild HFrEF
Mechanism of
action
Canonical
Cardiosphere-derived cells (CDCs)
First described by RR Smith et al., Circulation 2007

7. Peak Ecc strain@12mos Thickening@12mos Thickness@12mos
*
*
*
CADUCEUS
Final 12 mo data (Malliaras et al., JACC 2014)

8. Mechanistic rationale for allogeneic therapy
Transplanted CDCs
Cell proliferation
Differentiation
New healthy tissue of
donor origin
Short-term engraftment
Secreted factors
New healthy tissue of
host origin
CDCs anti-inflammatory, immunomodulatory and evanescent1-6
1. I. Chimenti et al, Circ Res 2010; 2. K. Malliaras et al, Circ 2012, 2013; 3. M.
Aminzadeh et al, Eur Heart J 2014; 4. E. Tseliou et al., Basic Res Cardiol 2014;
5. L. Lauden et al, Circ Res 2013; 6. E. Marbán, Mayo Clin Proc 2014

9. Deconstruction: follow the data
1. Autologous therapy (2004)
2. Recognition of durable benefits despite cell
transience (2010)
Allogeneic paradigm (2012)

10. Cell Type Cardiac stromal cells
Characteristics CD105+, CD45-; secreted SDF-1
Clinical Trials CADUCEUS-completed-autologous phase 1. Twenty-five patient study
showed regeneration in CDC-treated post-MI subjects with mild HFrEF
ALLSTAR-phase 1&2b study of allogeneic CDCs post-MI with mild HFrEF
DYNAMIC- phase 2a study of allogeneic CDCs in patients with advanced
HFrEF
HOPE, HOPE-2- phase1-2+ studies of allogeneic CDCs in patients with
Duchenne muscular dystrophy
Mechanism of
action
Paracrine effects
§ Promote cardiomyomyogenesis
§ Prevent cardiomyocyte apoptosis
§ Anti-fibrotic
§ Anti-inflammatory
Cardiosphere-derived cells (CDCs)

11. • Duchenne muscular
dystrophy
• X-linked recessive
disorder
• Skeletal myopathy
• Cardiomyopathy Cellular
[Ca2+]
Dystrophin
deficiency
Inflammation
Oxidative
stress
Myocyte loss
Fibrosis
Cell membrane
damage
Mitochondrial
inefficiency/loss

12. Duchenne cardiomyopathy:
Progressive increase in cardiac scar with patient age
Animesh Tandon et al. J Am Heart Assoc 2015;4:e001338

13. 0
200
400
600
800
1000
3 4 5 6 7 8 9 10 11 12
CTL(WT)
Mdx+CDC
Mdx+Vehicle
Functional improvement in mdx mice with
CDC treatment and repeat dosing
30
40
50
60
70
80
Baseline Wk3 M2 M3 Wk3 M2 M3
Mdx+CDC
Mdx+Vehicle
*** * *
* *
1st injection 2nd
injection
EF(%)
Global LV function
Distance(m)
Mdx+Vehicle
Mdx+CDC
Weeks
Exercise capacity
* *
*
*
*
*
*
*
*
*

14. First trial of CDCs in Duchenne patients
Halt cardiomyOPathy progrEssion in Duchenne:
HOPE-Duchenne trial
• DMD patients age 12+ with >4 segments of scar
by MRI
• N=25, 1:1 randomization to standard of care or
multivessel infusion of alloCDCs
• Endpoints: safety, scar by MRI, performance of
upper limb (PUL)

15. HOPE: reduced cardiac scar and improved PUL
M. Taylor et al., Neurology 2019

16. Marbán, Nature BME 2018
Exosomes: necessary and sufficient for CDC effects

17. IV CDCs work in mdx mice
R. Rogers et al. JCI Insight, 2019
Protocol Increased EF
Increased exercise capacity Decreased fibrosis

18. Deconstructing regenerative medicine
1. Autologous cell therapy (2004)
2. Recognition of durable benefits despite cell transience (2010)
Allogeneic paradigm (2012)
3. Identification of exosomes as mediators (2014)
Intravenous cell delivery in humans (2018-)

19. Follow-on trial of CDCs in Duchenne patients
HOPE-2 trial (active)
• DMD patients age 10+ with reduced arm strength
by performance of upper limb (PUL) testing
• N=40, 1:1 randomization to IV placebo or IV
alloCDCs (4 doses at 3-month intervals)
• Efficacy endpoints
– Primary: performance of upper limb (PUL)
– Secondary: regional function by cardiac MRI

20. HOPE-2
data
C. McDonald,
E. Marbán et al,
Lancet 2022
Performance of the upper limb (PUL) Cardiac function (LVEF) by MRI

21. Cell Type Cardiac stromal cells
Characteristics CD105+, CD45-; secreted SDF-1, exosomes
Clinical Trials CADUCEUS-completed-autologous phase 1. Twenty-five patient study
showed regeneration in CDC-treated post-MI subjects with mild HFrEF
ALLSTAR-phase 1&2b study of allogeneic CDCs post-MI with mild HFrEF
DYNAMIC- phase 2a study of allogeneic CDCs in patients with advanced
HFrEF
HOPE-2, -3 (recruiting)- IV allo CDCs for DMD cardiomyopathy
Mechanism of
action
Paracrine effects mediated by exosomes
§ Promote cardiomyomyogenesis
§ Prevent cardiomyocyte apoptosis
§ Anti-fibrotic
§ Anti-inflammatory
Cardiosphere-derived cells (CDCs)
First described by RR Smith et al., Circulation 2007; methods
and bioactivity reproduced by >75 labs worldwide

22. Deconstructing regenerative medicine
1. Autologous cell therapy (2004)
2. Recognition of durable benefits despite cell transience (2010)
Allogeneic paradigm (2012)
3. Identification of exosomes as mediators (2014)
Intravenous cell delivery in humans (2018-)
4. Mining of exosome contents identifies defined factors (2014-)

23. Exosomes: defined contents as next-gen TCs
miR-181b
miR-146a
Y RNA fragment
Cardioprotection
↑ PKCδ
↑ IL-10
↓ TRAF-6
G. DeCouto et al.,
Circ 2017
A. Ibrahim et al.,
Stem Cell Reports 2014
E. Marbán, Nature Biomed. Eng. 2018

24. miRs are minority of exosomal RNA
33.71
18.46
7.60
7.07
2.13
0.04
21.22
3.99
5.78
CDC-exo
85.70
0.92
2.24
0.67
1.65
0.29
4.98
1.66 1.89
NHDF-exo
L. Cambier et al., EMBO Mol Med 2017

25. Plentiful small Y RNA EV-YF1 is bioactive
0
5
10
15
20
25
Ys Yb
Relative
mRNA
IL10
expresssion
0
10
20
30
40
50
60
70
80
90
Ys Yb
IL10
concentration
(pg/ml)
24h
48h
72h
L. Cambier et al., EMBO Mol Med 2017
0
50000
100000
150000
200000
250000
300000
350000
400000
Yb Yc Yd
Nb
of
counts
Y RNA abundance Alignment reveals EV-YF1 homology to human Y-RNA4
dG=-14.00 kcal/mol
Predicted structure
**
****
****
IL-10 transcript (left) and secreted protein (right) in macrophages

26. A-M Yu et al., Pharmacological Reviews 2020
Properties of FDA-approved noncoding RNA drugs
All:
• 18-30 nucleotides long
• chemically-modified for
potency, stability and decreased
immunogenicity
• known mechanisms and
molecular targets (e.g., siRNA)

27. Reductionist engineering of a new ncRNA drug from EV-YF1
TY1: bioinspired 24 nt mutant LNA-modified small Y RNA
- New chemical entity
- Suppresses inflammation, fibrosis and hypertrophy
- Not targeted (i.e. not siRNA, aptamer etc)
- Mechanism?
Gene expression in transduced macrophages

28. Canonical full-length Y RNA mechanism
But, in TY1:
• Ro60 binding site is
disrupted
• La binding site is absent
• biotinylated TY1 does
not pull down Ro or La
Valkov N, Das S. Adv Exp Med Biol. 2020

29. TY1 associates with nucleoproteins
and uniquely with nuclear basket protein TPR
C
t
r
l
S
c
r
a
m
b
l
e
T
Y
1
0
10
20
30
TPR
(ng/ml)
C
t
r
l
S
c
r
a
m
b
l
e
T
Y
1
0
1
2
3
TPR
(peptide
hits)
Ran-GTPase
0
1
2
3
4
Septin7
Septin8
Septin9
Septin11
CRK
PRKAR1A
PPP1CB
PEBP1
0
1
2
3
4
AKAP12
TPR
NUP98
USP14
BUB3
SAE1
CUL4B
USP5
PRKAR1A
Nucleopore/Ubiquitination
Complex
mRNA-binding
0
1
2
3
4
5
CSDE1
G3BP1
PCMT1
TARDBP
PCNA
RECQL
EIF3L
EIF3CL
EIF3E
FXR1
EIF3J
NME2
TCEA2
CRIP2
Peptide hits
HFpEF: prominent
inflammation,fibrosis
and hypertrophy
TY1-
nucleoprotein
interaction
Altered
mRNA
transport
Inflammation
Fibrosis
Hypertrophy
Working hypothesis:

30. TY1 effective in 2-hit HFpEF mice
Physiology and serum biomarkers
mRNAs in control and HFpEF hearts

31. • Duchenne muscular dystrophy
• Scleroderma/ systemic sclerosis
What about other diseases of fibrosis and inflammation?

32. TY1 in mdx model of Duchenne muscular dystrophy
TY1 given after
disease established
improves EF,
exercise tolerance,
muscle force and
fibrosis

33. • Autoimmune disease affecting mostly
women 30-50 years of age
• ~60,000 new cases/year in USA
• Scar tissue deposits thicken and tighten
the skin, and often affect the heart and
lungs (systemic sclerosis)
• 28.3% ten-year mortality
• Incurable, no specific drugs or therapies
Scleroderma/ systemic sclerosis

34. TY1 works in bleomycin model of scleroderma
TY1 given after
disease
established
normalizes skin
thickness
… and cardiac
fibrosis

35. Next steps to the clinic
• Optimize dosing and formulation
• Clinical trial-grade, scalable manufacturing
• Long-term tox studies
• Draft clinical protocol
• Regulatory filings

36. Deconstructing regenerative medicine
1. Autologous cell therapy (2004)
2. Recognition of durable benefits despite cell transience (2010)
Allogeneic paradigm (2012)
3. Identification of exosomes as mediators (2014)
Intravenous cell delivery in humans (2018-)
4. Mining of exosome contents identifies defined factors (2014-)
5. Bioinspired new chemical entities (2021-)
Next-gen therapeutics

37. Acknowledgments
Smidt Heart Institute, Cedars-Sinai
Ahmed Ibrahim
Kazutaka Miyamoto
Alessandra Ciullo
Russell Rogers
Alice Rannou
Xaviar Jones
Funding
NIH
California Institute for
Regenerative Medicine
Department of Defense
Special appreciation to:
Jenny Van Eyk, Romain Gallet, Linda Cambier, Joshua Goldhaber,
Jae Cho, Thassio Ribeiro, Alice Rannou, James Dawkins, David
Lefer & Eugenio Cingolani

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