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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
risk factors like cholesterol & triglycerides.
(thread)
A thread 10+ years in the making.
This one starts in 2009 when I was a postdoc in @skathire lab @MassGeneralNews/ @broadinstitute studying
risk factors like cholesterol & triglycerides.(thread)
2/
Inspired by work from Boileau/Seidah/Breslow/Horton/Hobbs/Cohen/et al.:
1. Activating PCSK9 mutations =
LDL cholesterol
2. Inactivating mutations =
LDL & heart disease
3. People with no PCSK9 =LDL & *healthy*!
We asked: are there more PCSK9’s to be discovered?
Inspired by work from Boileau/Seidah/Breslow/Horton/Hobbs/Cohen/et al.:
1. Activating PCSK9 mutations =
LDL cholesterol2. Inactivating mutations =
LDL & heart disease3. People with no PCSK9 =
LDL & *healthy*!We asked: are there more PCSK9’s to be discovered?
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”:
LDL & triglycerides (TG) https://www.nytimes.com/2017/05/24/health/heart-drugs-gene-mutations.html
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”:
LDL & triglycerides (TG) 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.
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”!
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
common variants near ANGPTL3 linked to TG & LDL.
Thus:
PCSK9 = safe LDL
ANGPTL3 = safe LDL & TG
= candidate drug targets.
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
common variants near ANGPTL3 linked to TG & LDL.Thus:
PCSK9 = safe LDLANGPTL3 = safe LDL & TG= candidate drug targets.
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 disease modeling.
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
disease modeling.
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.
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.
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%PCSK9 in blood
≈40%cholesterol
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%
PCSK9 in blood≈40%
cholesterol
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 disease. https://www.sciencedaily.com/releases/2014/06/140610205512.htm
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
disease. 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 PCSK9.
Daniel Anderson’s lab @MIT used lipid nanoparticles (LNPs) to deliver Cas9 into mouse liver to PCSK9.
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
PCSK9.Daniel Anderson’s lab @MIT used lipid nanoparticles (LNPs) to deliver Cas9 into mouse liver to
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 PCSK9.
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
PCSK9.
14/
Around this time (2016-17), @skathire & I teamed up again, showing people with an inactivating mutation in ANGPTL3 to be protected from disease.
Independently shown by @Regeneron Genetics Center.
PCSK9 = safe LDL & risk
ANGPTL3 = safe LDL & TG & risk
Around this time (2016-17), @skathire & I teamed up again, showing people with an inactivating mutation in ANGPTL3 to be protected from
disease.Independently shown by @Regeneron Genetics Center.
PCSK9 = safe LDL & riskANGPTL3 = safe LDL & TG & risk
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 CT and AG changes in the genome—more precise, more efficient, and safer than Cas9.
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
T and AG changes in the genome—more precise, more efficient, and safer than Cas9.
16/
Alex Chadwick in my lab @PennMedicine used base editing in mouse liver to efficiently introduce nonsense mutations into PCSK9 or ANGPTL3.
WithANGPTL3 in mice with high cholesterol, she observed:
>50%triglycerides
>50%LDL
A double whammy!
https://pubmed.ncbi.nlm.nih.gov/29483174/
Alex Chadwick in my lab @PennMedicine used base editing in mouse liver to efficiently introduce nonsense mutations into PCSK9 or ANGPTL3.
With
ANGPTL3 in mice with high cholesterol, she observed:>50%
triglycerides>50%
LDLA double whammy!
https://pubmed.ncbi.nlm.nih.gov/29483174/
17/
PCSK9 antibodies were shown to reduce 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 disease—1-shot therapies with possibly lifelong protection. https://www.nature.com/articles/d41586-018-02482-4
PCSK9 antibodies were shown to reduce
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
disease—1-shot therapies with possibly lifelong protection. https://www.nature.com/articles/d41586-018-02482-4
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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
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
19/
Today @VerveTx announced new data from non-human primates in which LNP-delivered adenine base editor PCSK9 or ANGPTL3 in the liver.
PCSK9:
67% editing
89%PCSK9 in blood
59%LDL
ANGPTL3:
60% editing
95%ANGPTL3 in blood
64%TG
19%LDL https://www.businesswire.com/news/home/20200627005005/en/Verve-Therapeutics-Presents-New-Data-Non-Human-Primates
Today @VerveTx announced new data from non-human primates in which LNP-delivered adenine base editor
PCSK9 or ANGPTL3 in the liver.PCSK9:
67% editing
89%
PCSK9 in blood59%
LDLANGPTL3:
60% editing
95%
ANGPTL3 in blood64%
TG19%
LDL 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/ from @sxbegle
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/ from @sxbegle
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 from @ginakolata
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 from @ginakolata
22/
Here are the non-human primate data presented by @skathire on behalf of @VerveTx in his keynote talk at #ISSCR2020.
Here are the non-human primate data presented by @skathire on behalf of @VerveTx in his keynote talk at #ISSCR2020.
