Thread by @kiranmusunuru, How to use #CRISPR gene editing to fight heart disease, the #1 [...]

How to use #CRISPR gene editing to fight heart disease, the #1 killer worldwide:

A thread 10+ years in the making.

This one starts in 2009 when I was a postdoc in @skathire lab @MassGeneralNews/ @broadinstitute studying

https://abs.twimg.com/emoji/v2/... draggable="false" alt="❤" title="Rotes Herz" aria-label="Emoji: Rotes Herz"> risk factors like cholesterol & triglycerides.

(thread)

How to use #CRISPR gene editing to fight heart disease, the #1 killer worldwide:A thread 10+ years in the making.This one starts in 2009 when I was a postdoc in @skathire lab @MassGeneralNews/ @broadinstitute studying https://abs.twimg.com/emoji/v2/... draggable=

risk factors like cholesterol & triglycerides.(thread)" title="How to use #CRISPR gene editing to fight heart disease, the #1 killer worldwide:A thread 10+ years in the making.This one starts in 2009 when I was a postdoc in @skathire lab @MassGeneralNews/ @broadinstitute studying https://abs.twimg.com/emoji/v2/... draggable="false" alt="❤" title="Rotes Herz" aria-label="Emoji: Rotes Herz"> risk factors like cholesterol & triglycerides.(thread)" class="img-responsive" style="max-width:100%;"/>

2/

Inspired by work from Boileau/Seidah/Breslow/Horton/Hobbs/Cohen/et al.:

1. Activating PCSK9 mutations =

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬆️" title="Pfeil nach oben" aria-label="Emoji: Pfeil nach oben">LDL cholesterol

2. Inactivating mutations =

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">LDL &

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">heart disease

3. People with no PCSK9 =

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">LDL & *healthy*!

We asked: are there more PCSK9’s to be discovered?

2/Inspired by work from Boileau/Seidah/Breslow/Horton/Hobbs/Cohen/et al.:1. Activating PCSK9 mutations = https://abs.twimg.com/emoji/v2/... draggable=

3/

We scoured the literature and found that Dr. Gustav Schonfeld @WUSTLendo—an Auschwitz survivor, an immigrant to the US, a foremost expert on lipid metabolism—had identified 4 *healthy* siblings with a “double whammy”:

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">LDL &

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">triglycerides (TG) https://www.nytimes.com/2017/05/24/health/heart-drugs-gene-mutations.html">https://www.nytimes.com/2017/05/2...

Rare Gene Mutations Inspire New Heart Drugs

Some people carry gene mutations that leave them nearly impervious to heart attacks. Learning how these mutations work has led to a novel experimental treatment.

https://www.nytimes.com/2017/05/24/health/heart-drugs-gene-mutations.html

4/

Schonfeld hadn’t been able to pinpoint the causal gene with linkage analysis.

@skathire & I realized that a new technology unveiled in 2009—exome sequencing—might find the gene.

Schonfeld had DNA samples sent to us, and we applied exome sequencing to 2 of the 4 siblings.

5/

With exome sequencing so new, 2 students in the lab— @jpirruccello & @DoGenetics—set up a pipeline from scratch.

On a wintry Saturday afternoon, the pipeline finally spit out the thrilling answer:

Each sibling had 2 nonsense mutations in ANGPTL3—natural gene “knockouts”!

6/

ANGPTL3 was known to be linked to TG in mice, but not LDL.

That same year, GWAS of 100,000 people by @skathire, me, & many colleagues

https://abs.twimg.com/emoji/v2/... draggable="false" alt="➡️" title="Pfeil nach rechts" aria-label="Emoji: Pfeil nach rechts"> common variants near ANGPTL3 linked to TG & LDL.

Thus:

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">PCSK9 = safe

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">LDL

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">ANGPTL3 = safe

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">LDL &

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">TG

= candidate drug targets.

6/ANGPTL3 was known to be linked to TG in mice, but not LDL.That same year, GWAS of 100,000 people by @skathire, me, & many colleagues https://abs.twimg.com/emoji/v2/... draggable=

7/

While at @MassGeneralNews, I met @JKeithJoung—a foremost expert on gene editing—and learned from him how to make and use zinc-finger nucleases (ZFNs) to edit cells.

As I was starting my own lab @HSCRB, I used ZFNs and then TALENs to edit stem cells for

https://abs.twimg.com/emoji/v2/... draggable="false" alt="❤️" title="Rotes Herz" aria-label="Emoji: Rotes Herz"> disease modeling.

7/While at @MassGeneralNews, I met @JKeithJoung—a foremost expert on gene editing—and learned from him how to make and use zinc-finger nucleases (ZFNs) to edit cells.As I was starting my own lab @HSCRB, I used ZFNs and then TALENs to edit stem cells for https://abs.twimg.com/emoji/v2/... draggable=

disease modeling." title="7/While at @MassGeneralNews, I met @JKeithJoung—a foremost expert on gene editing—and learned from him how to make and use zinc-finger nucleases (ZFNs) to edit cells.As I was starting my own lab @HSCRB, I used ZFNs and then TALENs to edit stem cells for https://abs.twimg.com/emoji/v2/... draggable="false" alt="❤️" title="Rotes Herz" aria-label="Emoji: Rotes Herz"> disease modeling." class="img-responsive" style="max-width:100%;"/>

8/

With a few years of work, we figured out how to use TALENs to edit a variety of genes in stem cells.

Then in January 2013 the work of Doudna/Charpentier/Zhang/Church/Kim/Joung/et al. established CRISPR-Cas9 as a new gene-editing tool.

You probably know that story.

9/

We wondered: was CRISPR just another gene-editing tool? Would it work as well as ZFNs & TALENs?

Qiurong Ding in my lab compared CRISPR-Cas9 & TALENs head-to-head across many loci in stem cells.

We were stunned: CRISPR-Cas9 blew TALENs out of the water. It wasn’t even close.

10/

Our immediate thoughts: if CRISPR works so well in cells in a dish, would it work in a living animal? Could it be therapy?

When Ding delivered CRISPR-Cas9 targeting PCSK9 into adult mouse liver via an adenoviral vector:

>50% editing
≈90%

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">PCSK9 in blood
≈40%

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">cholesterol

10/Our immediate thoughts: if CRISPR works so well in cells in a dish, would it work in a living animal? Could it be therapy?When Ding delivered CRISPR-Cas9 targeting PCSK9 into adult mouse liver via an adenoviral vector:>50% editing≈90%https://abs.twimg.com/emoji/v2/... draggable=

PCSK9 in blood≈40%https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">cholesterol" title="10/Our immediate thoughts: if CRISPR works so well in cells in a dish, would it work in a living animal? Could it be therapy?When Ding delivered CRISPR-Cas9 targeting PCSK9 into adult mouse liver via an adenoviral vector:>50% editing≈90%https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">PCSK9 in blood≈40%https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">cholesterol" class="img-responsive" style="max-width:100%;"/>

11/

Subsequent work has shown PCSK9 editing persists for months to years in animal models—and might be permanent in human patients.

A one-and-done gene-editing therapy would be a new approach to reducing the 18 million deaths/year from

https://abs.twimg.com/emoji/v2/... draggable="false" alt="❤" title="Rotes Herz" aria-label="Emoji: Rotes Herz"> disease. https://www.sciencedaily.com/releases/2014/06/140610205512.htm">https://www.sciencedaily.com/releases/...

A shot against heart attacks? Scientists use genome-editing approach to mimic vaccination

A 'genome-editing' approach has been developed for permanently reducing cholesterol levels in mice through a single injection, a development with the potential to reduce the risk of heart attacks in...

https://www.sciencedaily.com/releases/2014/06/140610205512.htm

12/

PCSK9 became a test gene for innovations in therapeutic gene editing.

The lab of @zhangf delivered a small Cas9 (SaCas9) via an AAV vector into mouse liver to

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten"> PCSK9.

Daniel Anderson’s lab @MIT used lipid nanoparticles (LNPs) to deliver Cas9 into mouse liver to

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten"> PCSK9.

12/PCSK9 became a test gene for innovations in therapeutic gene editing.The lab of @zhangf delivered a small Cas9 (SaCas9) via an AAV vector into mouse liver to https://abs.twimg.com/emoji/v2/... draggable=

PCSK9.Daniel Anderson’s lab @MIT used lipid nanoparticles (LNPs) to deliver Cas9 into mouse liver to https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten"> PCSK9." title="12/PCSK9 became a test gene for innovations in therapeutic gene editing.The lab of @zhangf delivered a small Cas9 (SaCas9) via an AAV vector into mouse liver to https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten"> PCSK9.Daniel Anderson’s lab @MIT used lipid nanoparticles (LNPs) to deliver Cas9 into mouse liver to https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten"> PCSK9.">

13/

Xiao Wang in my lab used @Yecuris liver-humanized mice—the mouse’s own liver replaced with transplanted human liver cells—to show that Cas9 edits human PCSK9 in human liver cells in vivo efficiently.

Conclusion: if we could get Cas9 into the human liver, it would

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten"> PCSK9.

13/Xiao Wang in my lab used @Yecuris liver-humanized mice—the mouse’s own liver replaced with transplanted human liver cells—to show that Cas9 edits human PCSK9 in human liver cells in vivo efficiently.Conclusion: if we could get Cas9 into the human liver, it would https://abs.twimg.com/emoji/v2/... draggable=

PCSK9." title="13/Xiao Wang in my lab used @Yecuris liver-humanized mice—the mouse’s own liver replaced with transplanted human liver cells—to show that Cas9 edits human PCSK9 in human liver cells in vivo efficiently.Conclusion: if we could get Cas9 into the human liver, it would https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten"> PCSK9." class="img-responsive" style="max-width:100%;"/>

14/

Around this time (2016-17), @skathire & I teamed up again, showing people with an inactivating mutation in ANGPTL3 to be protected from

https://abs.twimg.com/emoji/v2/... draggable="false" alt="❤️" title="Rotes Herz" aria-label="Emoji: Rotes Herz"> disease.

Independently shown by @Regeneron Genetics Center.

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">PCSK9 = safe

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">LDL &

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">

https://abs.twimg.com/emoji/v2/... draggable="false" alt="❤️" title="Rotes Herz" aria-label="Emoji: Rotes Herz">risk

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">ANGPTL3 = safe

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">LDL &

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">TG &

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">

https://abs.twimg.com/emoji/v2/... draggable="false" alt="❤️" title="Rotes Herz" aria-label="Emoji: Rotes Herz">risk

14/Around this time (2016-17), @skathire & I teamed up again, showing people with an inactivating mutation in ANGPTL3 to be protected from https://abs.twimg.com/emoji/v2/... draggable=

15/

The next big innovation in gene editing was the development of cytosine and adenine base editors by the lab of @davidrliu, reported in 2016 & 2017.

These base editors allow for specific C

https://abs.twimg.com/emoji/v2/... draggable="false" alt="➡️" title="Pfeil nach rechts" aria-label="Emoji: Pfeil nach rechts">T and A

https://abs.twimg.com/emoji/v2/... draggable="false" alt="➡️" title="Pfeil nach rechts" aria-label="Emoji: Pfeil nach rechts">G changes in the genome—more precise, more efficient, and safer than Cas9.

15/The next big innovation in gene editing was the development of cytosine and adenine base editors by the lab of @davidrliu, reported in 2016 & 2017.These base editors allow for specific Chttps://abs.twimg.com/emoji/v2/... draggable=

T and Ahttps://abs.twimg.com/emoji/v2/... draggable="false" alt="➡️" title="Pfeil nach rechts" aria-label="Emoji: Pfeil nach rechts">G changes in the genome—more precise, more efficient, and safer than Cas9." title="15/The next big innovation in gene editing was the development of cytosine and adenine base editors by the lab of @davidrliu, reported in 2016 & 2017.These base editors allow for specific Chttps://abs.twimg.com/emoji/v2/... draggable="false" alt="➡️" title="Pfeil nach rechts" aria-label="Emoji: Pfeil nach rechts">T and Ahttps://abs.twimg.com/emoji/v2/... draggable="false" alt="➡️" title="Pfeil nach rechts" aria-label="Emoji: Pfeil nach rechts">G changes in the genome—more precise, more efficient, and safer than Cas9." class="img-responsive" style="max-width:100%;"/>

16/

Alex Chadwick in my lab @PennMedicine used base editing in mouse liver to efficiently introduce nonsense mutations into PCSK9 or ANGPTL3.

With

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">ANGPTL3 in mice with high cholesterol, she observed:

>50%

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">triglycerides
>50%

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">LDL

A double whammy!

https://pubmed.ncbi.nlm.nih.gov/29483174/ ">https://pubmed.ncbi.nlm.nih.gov/29483174/...

16/Alex Chadwick in my lab @PennMedicine used base editing in mouse liver to efficiently introduce nonsense mutations into PCSK9 or ANGPTL3.With https://abs.twimg.com/emoji/v2/... draggable=

ANGPTL3 in mice with high cholesterol, she observed:>50%https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">triglycerides>50%https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">LDLA double whammy! https://pubmed.ncbi.nlm.nih.gov/29483174/..." title="16/Alex Chadwick in my lab @PennMedicine used base editing in mouse liver to efficiently introduce nonsense mutations into PCSK9 or ANGPTL3.With https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">ANGPTL3 in mice with high cholesterol, she observed:>50%https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">triglycerides>50%https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">LDLA double whammy! https://pubmed.ncbi.nlm.nih.gov/29483174/..." class="img-responsive" style="max-width:100%;"/>

17/

PCSK9 antibodies were shown to reduce

https://abs.twimg.com/emoji/v2/... draggable="false" alt="❤️" title="Rotes Herz" aria-label="Emoji: Rotes Herz"> disease but have to be injected every few weeks.

It was now clear that gene/base editing of PCSK9 or ANGPTL3 was also a viable approach to reduce

https://abs.twimg.com/emoji/v2/... draggable="false" alt="❤️" title="Rotes Herz" aria-label="Emoji: Rotes Herz"> disease—1-shot therapies with possibly lifelong protection. https://www.nature.com/articles/d41586-018-02482-4">https://www.nature.com/articles/...

A CRISPR edit for heart disease

A one-off injection to reduce the risk of cardiovascular disease is now a prospect thanks to advances in gene editing.

https://www.nature.com/articles/d41586-018-02482-4

18/

The challenge now is to translate the gene editing to live human patients.

In 2018, @skathire, @JKeithJoung, & I were the scientific co-founders of @VerveTx, for which I serve as senior scientific advisor and where I’ve been on sabbatical this year. https://www.inquirer.com/health/crispr-verve-cholesterol-gene-upenn-dupont-20190611.html">https://www.inquirer.com/health/cr...

10-year-old’s cholesterol was over 800. Can CRISPR fix the problem?

Verve Therapeutics is considering a half-dozen candidate genes that could be edited with the CRISPR technique in order to sharply reduce a patient’s levels of cholesterol or triglycerides.

https://www.inquirer.com/health/crispr-verve-cholesterol-gene-upenn-dupont-20190611.html

19/

Today @VerveTx announced new data from non-human primates in which LNP-delivered adenine base editor

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten"> PCSK9 or ANGPTL3 in the liver.

PCSK9:
67% editing
89%

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇️" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">PCSK9 in blood
59%

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">LDL

ANGPTL3:
60% editing
95%

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">ANGPTL3 in blood
64%

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">TG
19%

https://abs.twimg.com/emoji/v2/... draggable="false" alt="⬇" title="Pfeil nach unten" aria-label="Emoji: Pfeil nach unten">LDL https://www.businesswire.com/news/home/20200627005005/en/Verve-Therapeutics-Presents-New-Data-Non-Human-Primates">https://www.businesswire.com/news/home...

Verve Therapeutics Presents New Data in Non-Human Primates Validating Gene Editing as a Treatment...

Verve Therapeutics, a next-generation cardiovascular company, today announced the presentation of new preclinical proof-of-concept data in non-human p

https://www.businesswire.com/news/home/20200627005005/en/Verve-Therapeutics-Presents-New-Data-Non-Human-Primates

20/

If these results can be reproduced in human patients, they’d rival or surpass the effects of all existing drugs that lower LDL or TG...

...but with a single treatment, rather than pills every day or injections every few weeks/months.

https://www.statnews.com/2020/06/27/crispr-base-editing-slashes-cholesterol-in-monkeys/">https://www.statnews.com/2020/06/2... from @sxbegle

CRISPR base editing slashes cholesterol levels in monkeys - STAT

When CRISPR “base editing” knocked out two cholesterol-associated genes in monkeys, the animals’ blood levels of LDL cholesterol and triglycerides plunged.

https://www.statnews.com/2020/06/27/crispr-base-editing-slashes-cholesterol-in-monkeys/

21/

This is an important step—in a long series of steps—to translate genetic discoveries into therapies to fight the preeminent health threat of the 21st century.

It’s been an enthralling 10 years—let’s see what the next 10 years bring!

https://www.nytimes.com/2020/06/27/health/heart-disease-gene-editing.html">https://www.nytimes.com/2020/06/2... from @ginakolata

A ‘Cure for Heart Disease’? A Single Shot Succeeds in Monkeys

A novel gene-editing experiment seems to have permanently reduced LDL and triglyceride levels in monkeys.

https://www.nytimes.com/2020/06/27/health/heart-disease-gene-editing.html

22/

Here are the non-human primate data presented by @skathire on behalf of @VerveTx in his keynote talk at #ISSCR2020.

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