Seminar about article. I only had two premises to discuss -not three-.

1. Universidad Pontificia Bolivariana
María Luisa Méndez Hernández
Third Semester 2022-1

2. A MI can be defined as a myocardial necrosis due to an
acute obstruction of a coronary artery -Irreversible
damage begins within 30 minutes of blockage-.
Symptoms of MI include chest discomfort with or
without dyspnea, nausea, and diaphoresis.
INTRODUCTION
A MI can diagnosed by:
-Symptoms of ischemia.
-ECG changes (ST/T changes or left bundle
branch block).
-Development of pathologic Q waves.
-Imaging evidence of new loss of
myocardium.
-Angiography evidence of intracoronary
thrombus.
MYOCARDIAL
INFARCTION
MI can be classified into 5 types:
1: MI caused by ischemia, due to a coronary event.
2: Ischemia due to ↑ O2 demand or ↓ supply.
3: Related to sudden cardiac death.
4a: Associated with coronary intervention.
4b: Associated with stent thrombosis.
5: Associated with CABG.

3. CRISPR-Cas9 is a tool used for editing parts of the genome by
removing, adding or altering sections of the DNA sequence.
INTRODUCTION CRISPR
The CRISPR system consists of two molecules:
- Cas9: An enzyme that acts as “molecular scissors” that can cut
DNA at a specific locations.
- Guide RNA (gRNA): A small piece of pre-designed RNA
sequence (aprox, 20 bases). gRNA makes sure that the Cas9
enzyme cuts at the right point in the genome.
Scientists developed this tool by adapting a similar gene editing
system that some bacteria have to respond to pathogens.
CRISPR-Cas9 has potential as a tool for treating medical
conditions that have a genetic component, such as cancer,
hepatitis B and high cholesterol.

4. First, we developed
CRISPR/Cas9 magnetoplexes
that magnetically guided
CRISPR/Cas9 system to the
heart for efficient in vivo
therapeutic gene targeting
during heart failures.
OBJECTIVES
#1 #2
We then demonstrated that
the in vivo gene targeting of
miR34a via these CRISPR/
Cas9 magnetoplexes in a
mouse model of myocardial
infarction significantly
improved cardiac repair and
regeneration to facilitate
improvements in cardiac
function.

5. 2.8 Immunocytochemistry 2.11
RNA isolation and quantitative reverse
transcription–polymerase chain reaction (qRT-PCR)
METHODOLOGY
Immunohistochemical analysis was
performed using cells and mouse heart
sections. This technique is usually used
for detection and visualization of
proteins or other antigens in cells, by
use of a specific primary antibody that
binds to it. It was used to identify the
proliferation of Cas9 magnetoplex-
treated cardiomyocytes post-H2O2
induction. By the recognition of PH3 -
which marks the active phases of
proliferation- and α-actinin expression.
- RNA isolation helps to separate RNA from tissues, DNA
or proteins; it’s based on liquid-phase separation.
Meanwhile, qRT-PCR enables detection of products
generated during the PCR process. It’s based on an
oligonucleotide probe that hybridizes within the target
sequence. Cleavage of the probe during PCR due to Taq
polymerase can be used to detect amplification of the
target-specific product. This technique was used to isolate
miRNA from cells; and then, the extracted miRNA was
reverse-transcribed. The purpose of using this method was
to examine the efficiency of the Cas9 magnetoplex-
mediated genome targeting; thus, it was used for analyzing
the miRNA present in the treated cells.

6. 2.12 2.15
This method involves electrophoresis and is
based on the random incorporation of chain-
terminating dideoxynucleotides by DNA
polymerase during in vitro DNA replication. It’s
used to study a small subset of genes linked to
a defined phenotype, confirm next-generation
sequencing variants, detect minor allele
fractions, or read contiguous sequences. In this
study, it was used to examine whether Cas9
magnetoplexes can cleave the target miR34a in
vivo. This analysis identified mutations caused
by NHEJ, confirming that miR34a was cleaved
in vivo.
METHODOLOGY
Western blot analysis
In this technique a mixture of proteins is
separated based on molecular weight, by
electrophoresis. These results are transferred to
a membrane producing a band for each protein.
The membrane is then incubated with labels
antibodies specific to the protein of interest. It
was used to analyze Notch1 and Pnuts in cells
treated with miR34a-Cas9 magnetoplexes. It
was, also, used to identify caspase-3 and actin
in cells treated with miR34a-Cas9
magnetoplexes. Thus, with this method the
efficiency of the Cas9 magnetoplex-mediated
genome targeting can be identified.
Sanger sequencing analysis

7. RESULTS
As the gel runs, shorter DNA
fragments will travel faster
through the pores of the gel
matrix than larger fragments.
Which means that they
introduced correctly the MNPs to
the CRISPR/Cas9 vector; there’s
more DNA.
They are showing the
results of proteins. If the
line is thicker, it means it has
more expression. The
results show that the
miR34a, after the
introduction of the
CRISPR/Cas9, had less
expression.
Actin is the test control. Here,
they are showing the results of
proteins. If the line is thicker, it
means it has more expression.
The results show that the
Notch1 and pnuts had more
expression, after the
introduction of the
CRISPR/Cas9 treatment.

8. RESULTS
Actin is the test control.
They are showing the results
of proteins. If the line is
thicker, it means it has more
expression. The results show
that the Caspase 3, after the
introduction of the
CRISPR/Cas9, had less
expression.
Here they are showing us an immunofluorescence, where the
fluorochromes are marking different regions at the cellular level. This
results show greater expression of proteins in post-MI heart treated
with miR34a-Cas9 magnetoplexes. This is because the miR34a
expression is accompanied by downregulation of several direct
targets, including Notch1, Sirt1, Pten, and Pnuts. Which means that
this proteins after eliminating miR34a began to show and increase
cell survival, cell growth, and DNA repair.

9. DISCUSSION
Heart failure has become an important health
issue worldwide. Impressive advances in
molecular cardiology over the past decades have
identified potential therapeutic goals for cardiac
therapy (Boon, et al; Bernardo, et al; Xiao, et al;
Wang, et al; Xu, et al)
Among the miRNAs, miR34a is upregulated over
time in aging heart, under cardiac injury, and in
patients with heart failure; moreover, knockout
of miR34a was shown to yield efficient protective
effects against cardiac failure after acute
myocardial infarction (Boon, et al; Bernardo, et
al; Yang, et al; Rokavec, et al)
✅ ✅

10. 2. Molecular biology is an area
that is intrinsically involved
with research. Which makes
it essential, nowadays,
because of the various
techniques that have been
created with it; which have
given medicine a path to
explore the various diseases
with genetic components and
a possible treatment for
them.
1. Molecular biology, with its
discoveries, has managed to
open doors that were
previously considered
impossible in the world of
medicine. By researching the
various cellular molecules, that
carry out essential biological
processes for the functioning
of the cell and its maintenance,
it has been possible to
generate various treatments
for diseases with genetic
components, which makes this
area of study one of the most
relevant in the current world.
CONCLUSIONS

11. In vivo therapeutic genome editing via CRISPR/Cas9 magnetoplexes for
myocardial infarction
What’s an MI?
A myocardial
necrosis due to an
acute obstruction of
a coronary artery.
What’s
CRISPR?
Is a tool used for
editing parts of
the genome by
removing, adding
or altering
sections of the
DNA sequence.
What are the
objectives in this
study?
What methods were
used in this study?
What were the
results?
Previous
Knowledge
- To develop MNPs
that guide CRISPR
to the heart for
therapeutic gene
targeting during
heart failures.
- In a mouse model
of MI this treatment
significantly
improved cardiac
repair and
regeneration.
-Immunocytochemistry.
-RNA isolation and qRT-
PCR.
-Western blot analysis.
-Sanger sequencing
analysis.
-The introduction of
the MNPs to the
CRISPR vector was
effective.
-The miR34a and
Caspase 3, after the
introduction of the
CRISPR/Cas9, had
less expression.
-Notch1 and pnuts
had more expression,
after the introduction
of the CRISPR/Cas9
treatment.
Discussion
Through the study
they could achieve
their objectives
and demonstrate
their premises
about the
importance of
investigating heart
failure and how
knockout of
miR34a was shown
to yield efficient
protective effects
against cardiac
failure after an MI.