In vivo (liver) human genome editing tests were conducted in the first clinical trial.

Scientists have edited the human genome before, but it is always carried out in cells outside the body.
Today, Sangamo Therapeutics, a biotech company, is recruiting participants to several clinical trials: non-functional enzymes in patients with haemophilia B, Hurler syndrome, or Hunter syndrome, which break double strands of DNA in their genomes and insert genes that encode functional enzymes into their genomes.
s the first time a person may have a new gene in their liver," Sandy Macrae, president and chief executive officer of Sangamo, told Scientist.
it is an honor and a responsibility to conduct these clinical trials.
sangamo will focus on haemophilia B, a severe hemorrhagic disease caused by defects or deficiencies in the F9 gene.
protein product of this gene, the clotting factor IX, is an enzyme essential for blood clotting.
two other clinical trials will focus on Heller syndrome and Hunter syndrome.
patients with both diseases lack a functional copy of two different genes.
these two genes encode enzymes that degrade complex polysaccharose enzymes involved in processes such as development and angiogenesics.
without these enzymes, specific polysaccharide types called glycosaminoglycan accumulate in cells, which can lead to neurological problems, cardiovascular disease and other symptoms.
In all three clinical trials, ZFN was cut at the target of the albumin-encoded gene in human liver cells, and a functional copy of the gene was then integrated into the genome of the liver cell through cogeneration.
the initial phase of a clinical trial involved six haemophilia B patients, each of who will receive intravenous injections of low-, medium- or high-dose SB-FIX drugs consisting of three adeno-related viruses (AAVs): two AAVs containing genes that produce ZFN and a third AAV containing functional F9 genes.
Macrae said the researchers expect F9 DNA to be integrated into the target of the albumin-coding gene in a small number of liver cells, but given the high level of albumin expression in the liver, coagulation factor IX levels in these patients can still increase.
Sangamo evaluated the drug's safety and determined its optimal dose, three haemophilia B patients will participate in the clinical trial as part of the optimal dose group.
Davidoff, a pediatric surgeon at St. Jude Children's Research Hospital in the United States, said haemophilia B is "the first disease that can be targeted because the physiological characteristics of the disease are very clear."
he explains, "If you're just replacing this missing or defective coagulation factor, you're actually going to cure these patients."
Davidoff is not involved in Sangamo's upcoming clinical trial, but he has led a clinical trial of hemophilia B male patients: the AAV virus delivers a functional copy of F9 to the liver.
in Davidoff's clinical trials, the gene box was not integrated into the patient's genome, but was present in liver cells as extragenome DNA called episomes.
, "Given the absence of integration, these add-ons may be lost if the target cells divide," explains Davidoff.
, even if F9 is not integrated into the genome, these male patients who received the treatment in Davidoff's clinical trials increased the expression of this coagulation factor, leading to improved clinical symptoms.
the earliest participants showed consistent circulatory coagulation factor IX levels over a period of more than 7 years.
, "It's a bit surprising that things have been stable," said Davidoff, a director of the U.S. Government.
part of the answer is that liver cells rarely divide without trauma or surgery.
"Macrae said, one advantage of Sangamo's strategy in current clinical trials is that it is "a lifelong solution."
once these functional copies of the ZFN-promoting F9 gene are integrated into the participants' liver cell genomes, the gene should be expressed steadily.
Paula Cannon, a gene therapy researcher at the University of Southern California's Keck School of Medicine, described the albumin-coding gene as a "safe harbor site" --- which experimentally identifies genomic site suitable for gene insertion and long-term expression.
Canon is not involved in the current clinical trials, but she has previously worked with Sangamo to integrate a gene into human cells using ZFN to make their offspring resistant to HIV infection.
Canon told Scientist that both ZFN and CRISPR/Cas9 genome editing systems are able to cut DNA in a site-specific way, but added that ZFN also has an advantage in developing clinical therapies.
, for example, there are limitations to CRISPR/Cas9, i.e. the wizard RNA (gRNA) that binds to the target sequence must be attached to an anchor sequence (usually containing two G bases).
, CRISPR/Cas9 "doesn't provide the high specificity you want," she said.
," Cannon adds, "ZFN is a little bit harder to develop," but the system developed by Sangamo "gives you a very specific and highly effective ZFN."
they were able to allow ZFN to cut any particular base.
"Cannon highlights ZFN's history and being approved by the U.S. Food and Drug Administration (FDA) as an experimental new drug.
ZFN has been successfully used to knock out a gene involved in HIV-invading human T cells, which do not trigger an immune response when injected into the body.
benefit of using ZFNs is that they are smaller than Cas9, which is required for CRISPR/Cas9 systems.
when editing cells outside the body, it doesn't matter," Cannon said.
but if you want to process tissue in your body, as in the current clinical trial, you'll look at a class of vectors called AAVs.
really hard to insert Cas9 into this type of vector because it's too big.
", however, some of the barriers to genome editing in the body are widespread.
Robert Doms, a pathologist at the University of Pennsylvania School of Medicine who was not involved in the current clinical trials, said, "Whether using ZFN or similar TALEN or CRISPR/Cas9 systems, they have shipping problems, which is how to transport the products of interest to specific cells rather than other cells."
no matter what you try to do, this is a challenge for gene therapy.
"For Sangamo's current clinical trials, it's fairly easy to transport any of these drugs to the liver because AAVs can easily invade liver cells through the blood."
other research teams have successfully transported AAVs into the eye, and Sangamo may soon optimize AAV deliveries to the brain, explains Macrae.
but his agreement to deliver the drug to the right organization is an immediate problem.
he said, "I believe the secret to future use and editing of applications lies in shipping."
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