Genetic defects are exceedingly common, which is not surprising considering just how many cells make up our bodies, including our reproductive cells. While most of these defects have no or only minor effects, some range from serious to fatal. One of these defects is in the CPS1 gene, with those affected facing a shortened lifespan along with intensive treatments and a liver transplant as the only real solution. This may now be changing, after the first successful genetic treatment of an infant with CPS1 deficiency.
Carbamoyl phosphate synthetase I (CPS1) is an enzyme that is crucial for breaking down the ammonia that is formed when proteins are broken down. If the body doesn’t produce enough of this enzyme in the liver, ammonia will accumulate in the blood, eventually reaching levels where it will affect primarily the nervous system. As an autosomal recessive metabolic disorder it requires both parents to be carriers, with the severity depending on the exact mutation.
In the case of the affected infant, KJ Muldoon, the CPS1 deficiency was severe with only a low-protein diet and ammonia-lowering (nitrogen scavenging) medication keeping the child alive while a search for a donor liver had begun. It is in this context that in a few months time a CRISPR-Cas9 therapy was developed that so far appears to fixing the faulty genes in the liver cells.
Despite its toxicity to living beings, ammonia (NH3) is an essential part of these same living beings, primarily in the form of amines (R-NH2), itself a rather indispensable part of amino acids, specifically the 22 proteinogenic amino acids from which proteins are formed. Just as ammonia is required for the amination process, so too is ammonia formed inside the body mostly as the result of transamination and deamination of these biogenic amines. This is a process that takes place primarily in the liver and involves the deamination of both the body’s own waste proteins as well as those from one’s diet.
Since only part of the ammonia can be reused for new amino acids, the rest has to be neutralized. Due to the toxicity of ammonia, blood levels have to be limited to <50 µmol/L or hyperammonemia will occur. This is where the urea cycle comes into play to maintain a healthy ammonia level.
The very first step of the urea cycle is the conversion of ammonia to carbamoyl phosphate:
Normally this is a very slow reaction, which is where the enzyme CSP1 comes into play as catalyst. In humans the gene for this enzyme is located on chromosome 2’s long arm, at locus 2q34. If there is a mutation in this gene that prevents it from working as a catalyst, ammonia levels in blood plasma will keep rising, eventually reaching levels where the nervous system is affected. In infants this is noticeable as lethargy, seizures and a lack of normal developmental milestones. Without treatment, developmental delay, intellectual disability or death affect 50% of babies.
Undoing A Mutation
When KJ was born on August 2024, it was noticed that he was lethargic, with stiff muscles and other worrisome symptoms. After a severe CPS1 deficiency was diagnosed via genome sequencing, KJ was hospitalized at only five months old. KJ’s only hope appeared to be a liver transplant and was put on the list for a donor organ, providing a slim hope at best. Meanwhile, a team of researchers started researching the cause of KJ’s CPS1 deficiency and the mutations behind it.
As described by Dr. Eric Topol in his summary of the (paywalled) paper by Gropman et al. in NEJM, both the father and mother were found to be carriers for CPS1 mutations, with the father carrying the truncating Q335X variant and the mother another (E714X). If either mutation could be corrected, the child would have one functional copy and theoretically be able to produce enough CPS1 to have a functional urea cycle without external assistance.
A complicating issue here is that despite the many reports of gene-editing with CRISPR the past years, there are various gradations, with what Dr. Topol refers to as CRISPR 1.0 through 3.0:
CRISPR 1.0: A CRISPR-Cas9 tool causes sufficient double-strand damage to disable the gene (knock-out). Crude and not relevant here. Also performed ex vivo.
CRISPR 2.0: Introduced single-strand cuts that allow for limited base editing, e.g. swapping A for a G.
CRISPR 3.0. Expands base editing to include multiple base pairs, both ex vivo and in vivo.
These methods have previously already been used ex vivo to create modified T-cells for CAR T-cell immunotherapy in the context of cancer treatments. In terms of in vivo treatments, there is the 2023 knocking out of PCSK9 liver protein to reduce bad cholesterol levels and the more recent base editing of the PiZ mutation responsible for liver and lung damage. There’s also ARCUS, which is a viral vector-based method of base editing that has seen use in fixing another urea cycle-related disorder.
Although only CRISPR 2.0 was needed here, what was unique in the case of KJ was that this would be the first fully personalized base editing therapy, applied in vivo and developed within the span of a mere six months.
Crossing All The Ts
K-abe base editor bound to target sequence of CPS1 (Credit: Gropman et al. NEJM, 2025)
With how experimental this gene therapy for KJ’s CPS1 disorder was, the researchers had to go through the entire gamut of tests, including on animal models. With a base editor developed to target the father’s Q335X mutation and rewrite it to the correct base pairs, mice were bred that had the same CSP1 mutation, in addition to testing on non-human primates, all to validate the approach and gain FDA approval.
The base editor’s goal was to rewrite the the wrong bases at the Q335X location on locus 2q34. A concern with any application of CRISPR is so-called off-target edits, but the safety review seems to have passed here without serious issues.
Starting with a very low dose, blood plasma ammonia levels were carefully monitored with no noticeable changes. Three weeks later the second, higher dose was injected, with reportedly positive effects on the ammonia levels. A third dose was injected a while later, though the results of this aren’t know yet. In the absence of a liver biopsy it is hard to say in how far this is a true cure, as reported so far is a reduced need for medications.
Per reports, KJ is however doing better, hitting developmental targets and got over two viral infections, without an ammonia crisis. Further injections of the treatment will likely administered with an mRNA approach rather than the (presumed) virus vector used so far due to immunity concerns with a virus vector. Open questions remain regarding how many cells have been truly edited in KJ’s liver and what the overall effectiveness is.
This leads us to cautiously welcome this news as a step forward in personalized gene-therapy, while realizing that the road ahead for both KJ and the rest of us is still full of unknowns and challenges. That said, one can only hope for KJ’s best possible progress and ideally serving as a beacon of hope for others afflicted by genetic disorders like CPS1 deficiency.
This articles is written by : Nermeen Nabil Khear Abdelmalak
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