Is it ever wise to edit wild-type alleles?

Abstract

The tremendous burden of lipid metabolism diseases, coupled with recent developments in human somatic gene editing, has motivated researchers to propose population-wide somatic gene editing of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) within the livers of otherwise healthy humans. The best characterized molecular function of PCSK9 is its ability to regulate plasma low density lipoprotein (LDL) levels through promoting LDL receptor degradation. Individuals with loss-of-function PCSK9 variants have lower levels of plasma LDL and reduced cardiovascular disease. Gain-of-function variants of PCSK9 are strongly associated with familial hypercholesterolemia (FH). A new therapeutic strategy delivers CRISPR/Cas9 specifically to liver cells to edit the wild-type alleles of PCSK9 with the goal of producing a loss-of-function allele. This direct somatic gene editing approach is being pursued despite the availability of FDA approved PCSK9 inhibitors that lower plasma LDL levels. Here, we discuss other characterized functions of PCSK9 including its role in infection and host immunity. We explore important factors that may have contributed to the evolutionary selection of PCSK9 in several vertebrates, including humans. Until such time that more fully understand the multiple biological roles of PCSK9, the ethics of permanently editing the gene locus in healthy, wild-type populations remains highly questionable.

Starting from a primordial single-celled organism, evolutionary processes have yielded an incredible diversity of creatures that include the great white shark, the giraffe, and the giant sequoia. Like these organisms, the sentient, upright human evolved by natural selection. The process of change and adaptability has been both random and guided by selection from evolutionary forces that may be difficult to elucidate or even know. Recent gene-editing technologies have enabled, for the first time, humans to directly edit gene variants known to cause disease. Somatic gene editing can target DNA in a specific population of cells (e.g. liver hepatocytes) without altering the individual’s reproductive cells, and thereby would not be inherited by future generations which is in contrast to germline gene editing approaches. While many human diseases that affect worldwide morbidity and mortality (cardiovascular disease, diabetes, obesity, cancer) are influenced by multiple genes, there are several cases where disease development is the direct result of alterations in single genes. In these specific cases, somatic gene editing might be applied to restore a gene sequence to a common variant (wild-type) thereby treating genetically inherited diseases and offering the potential to profoundly improve the health of these individuals.

What does it mean to be “wild-type”?

The textbook definition of the term wild type, “refers to a strain, organism, or gene that is most prevalent in the ‘wild’ population of the organism with respect to genotype and phenotype” ^1,2. “Wild-type” encompasses many evolutionary modifications, or variations, of a gene. A variation from of a wild-type allele is often referred to as a mutant allele¹. A wild-type allele frequently has a positive connotation, while the term “mutant” incites a negative undertone. An obvious example of a mutant allele would be a disease-causing gene variant. Ultimately, however, wild-type and mutant alleles are not necessarily synonymous with a desirable phenotype and undesirable phenotype.

In the case where a single gene variant has been linked to a disease, a directed edit can be made to change the gene into a common, wild-type variant. For example, one hypothetical candidate for somatic gene editing is cystic fibrosis transmembrane conductance regulator (CFTR) gene ³. A person who is homozygous for the mutant allele will develop cystic fibrosis and will have a life expectancy of approximately forty years ⁴. The genetic sequence of the CFTR gene can be directly edited to produce a more common wild-type variant, thereby rescuing CFTR function, and lessening disease risk ⁵. Editing the CFTR locus would be an example of disease treatment by editing a rare, disease-causing mutant variant to result in a wild-type variant. In this article, we are going to discuss the ethics of using somatic gene editing to target a wild-type allele with the goal of producing a mutant allele.

The National Academies of Sciences, Engineering, and Medicine formalized recommendations for the use of human genome editing and have divided the ethical considerations into three classifications: disease treatment, disease prevention, and enhancement ¹. Both disease treatment and prevention involve the modification of a gene variant that is known to cause harm to a variant that is not associated with disease. The non-disease state is commonly referred to as “normal.” “Normal,” or a natural state, does not encompass one ideal condition, but rather represents a range of gene variants that are prevalent in the population. In contrast, enhancement is when a gene variant may be altered to produce an advantageous phenotype beyond restoring or maintaining an individual’s health. While there is a strong ethical justification ⁶ to genetically edit a disease-causing variant, the consensus among international regulatory agencies is that it is not appropriate to gene edit any allele with the goal to “enhance” an individual ¹. The National Academies specifically pointed out the challenge of distinguishing between therapy and enhancement regarding treatments that modify plasma lipids:

“[…] genome editing to lower the cholesterol level of a patient with severe coronary artery disease would likely be viewed as a therapy, and genome editing of a sibling of the patient with high cholesterol who also had other risk factors for coronary artery disease might be viewed as a preventive measure, genome editing to lower the cholesterol of a healthy 21-year-old child of the patient to reduce disease risk below what is average or “normal” in the general population might be viewed as approaching the line between prevention and enhancement. Interventions thus can be viewed as falling on a therapy–prevention–enhancement spectrum, although the boundaries between the three categories are still open to debate and will likely vary with the specifics of the intervention” ¹.

Is it wise to create a PCSK9 loss-of-function variant?

Familial hypercholesterolemia (FH) is a genetic disorder caused by loss-of-function mutations in the low-density-lipoprotein (LDL) receptor, apolipoprotein B, and/or gain-of-function mutations in proprotein convertase subtilisin/Kexin type 9 (PCSK9) ⁷. Mutations in these genes affect LDL clearance and cause high levels of plasma LDL cholesterol. A person who is homozygous for a loss-of-function allele of the LDL receptor will develop severe cardiovascular disease during adolescence and typically experience significant morbidity and mortality with a fifty percent chance of dying from a cardiovascular event by age 32 ^8,9. Heterozygous FH affects between 1 in 250 people ⁷ and is associated with an increased risk of a severe cardiovascular event ¹⁰. PCSK9 inhibitors are FDA approved lipid-lowering therapies for individuals carrying ApoB mutations, heterozygous for the LDL receptor loss-of-function alleles or that have gain-of-function alleles of PCSK9. These therapeutics include PCSK9 monoclonal antibodies, RNA-based therapies, and PCSK9 vaccines ^11–14. Small molecule inhibitors of PCSK9 are also under development and showing promise ¹⁵. The current widespread use of these PCSK9 biologics is limited by their cost and side effects that can result in poor patient compliance ¹⁴. These issues have motivated some scientists and pharmaceutical manufacturers ¹⁶ to propose targeting the PCSK9 locus for treatment of FH by somatic gene editing (excluding individuals homozygous for LDL receptor loss-of-function alleles).

PCSK9 is a multi-functional protein that is expressed in the liver, intestine, kidney, and adrenal tissue. However, the liver is the primary source of plasma PCSK9. PCSK9 alleles are strongly associated with FH ^17,18. LDL-receptor degradation is the best characterized function of PCSK9, where PCSK9 circulates in the plasma, binds the cell surface LDL-receptors, and targets the LDL-receptor to the lysosome for degradation ¹⁹. PCSK9 gain-of-function mutations cause a reduction in LDL-receptors on the plasma membrane of hepatocytes resulting in higher levels of circulating LDL ¹⁸. In contrast, people with complete loss-of-function variants of PCSK9 show a substantial reduction in circulating LDL particle number and cardiovascular disease risk due to higher numbers of LDL-receptors on the cell surface ²⁰. Individuals with PCSK9 loss-of-function variants have not been found to have other disease risks nor overt developmental abnormalities ^21,22. Given that cardiovascular disease is one of the top sources of worldwide mortality, and individuals with PCSK9 loss-of-function variants “seem” fine, why not inactivate wild-type alleles of PCSK9 at the population level to mitigate the burden of cardiovascular disease?

Verve Therapeutics proposes to use a targeted somatic genome editing to create loss-of-function alleles in the PCSK9 gene locus as a treatment for individuals who are heterozygous for the loss-of-function alleles of the LDL receptor. The PCSK9 gene editing strategy is not designed for patients with homozygous loss-of-function LDL receptor alleles because there are no functional LDL receptors to upregulate as a result of PCSK9 loss. Verve Therapeutics indicates that this strategy can be expanded to “address larger populations of patients with or at risk for atherosclerotic cardiovascular disease” ¹⁶. This could include individuals whose lifestyle, diet, BMI and/or other nongenetic factors that put them at risk for disease. One argument for such an approach is that the cost and inconvenience of current therapies justify a somatic gene-editing-based approach of PCSK9 as a proposed treatment for the dyslipidemias associated with highly prevalent metabolic diseases. However, proposing to edit wild-type alleles obscures the boundary between therapy and enhancement because the intent of the modification would be to optimize and provide a beneficial effect to the individual rather than to modify a disease-causing allele. Editing humans based on the statistic that individuals could, perhaps, be sick in the future after years of physical inactivity and consuming a Western diet ²³, muddles the distinction between therapy and enhancement. Moreover, it assumes that the only function of PCSK9 is to modulate levels of the LDL receptor.

Genetic diversity and domestication

PCSK9 is present in most vertebrates including humans, primates, and rodents, yet has been lost in pigs, cows, camels, horses, dogs, and cats ²⁴. It is immediately apparent that loss of PCSK9 occurred in domesticated animals where humans were likely controlling food sources and availability. Restricting food availability can remove a potentially powerful selective force that could maintain a gene in the genome. Gene loss during domestication ²⁴, and a decrease of gene expression diversity has been observed in domesticated animals ²⁵. Often, domesticated species are, in fact, not very good at surviving in the wild without human cultivation.

So, what is the selective pressure maintaining PCSK9 in the human genome? Ancient humans were hunter-gatherers likely living on hunted fowl and berries ²⁶. Denisovan and Neanderthal sequencing revealed unique PCSK9 mutations predicted to result in loss-of-function alleles ²⁶. Loss-of-function variants of PCSK9 may have been tolerated if dietary lipid was in abundance, but might have been a disadvantageous to human ancestors who had to survive long periods of lipid-poor meals ^13,26. Our fundamental understanding of genetic selection stipulates that while a few rare families have loss-of-function alleles of PCSK9 which serve to protect them from cardiovascular disease, the wild-type protein has important functions precisely because it has remained functional under selective pressure for over 350 million years throughout vertebrate evolution from fishes to humans. As we will describe below, there is evidence that PCSK9 influences immunity either directly through its effects on the surface-localization of the LDL-receptor and/or by its ability to modulate plasma lipids. These functions may underlie the selective advantages of maintaining this gene in the genome and should be considered before we rush to eliminate PCSK9 activity as a protection against cardiovascular disease. Although we are not providing a comprehensive review of the literature of PCSK9, here we highlight evidence for other potentially critical functions of PCSK9.

Important roles of PCSK9

PCSK9 levels can increase during viral infection, specifically in HIV and hepatitis C-infected individuals ²⁷. The life cycle of hepatitis C virus (HCV) relies on host lipoprotein machineries as a method to evade recognition by host immunity ²⁸. HCV circulates as a highly-lipidated particle, much like a lipoprotein, to gain entry into hepatocytes ²⁷. During infection, PCSK9 upregulation may help to clear infection by increasing degradation of the LDL receptor, thus leaving lipid-virus particles in circulation rather than promoting viral uptake, replication, and assembly ^27,29. Viral infection is not the only immunological challenge associated with changes in PCSK9 expression. High levels of PCSK9 have been recorded in patients with a lower respiratory tract infection and in bacteremia caused by Streptococcus pneumoniae ³⁰. PCSK9 is also upregulated during sepsis, and the degree of PCSK9 upregulation is positively correlated with the likelihood of survival ^30,31. Serum lipids (cholesterol, triglycerides, and LDL) increase during pregnancy ³². Additionally, there is evidence for elevated levels of serum PCSK9 during pregnancy compared to the non-pregnant state ³³, and PCSK9 levels continue to rise as normal pregnancy progresses ³⁴. It is reasonable to hypothesize that PCSK9 increases lipid availability during pregnancy to provide lipid to the developing fetus, in a similar way that pregnancy-associated insulin resistance is thought to provide additional glucose to the developing fetus ³⁵.

PCSK9 levels are a useful biomarker for toxoplasmosis screening which is the disease caused by Toxoplasma gondii. Primary infection in pregnant woman can negatively impact the developing fetus ³⁶. In studies exploring the immunological response to T. gondii in pregnant women and women who have had abortions revealed that patients infected with T. gondii had increases in both PCSK9 and blood lipids (HDL, VLDL, and triglycerides) and a decrease in LDL ³⁶. Researchers hypothesize that the difference in patient lipid profiles is due to the dependence of T. gondii on host metabolic pathways for the intracellular development of the parasite ³⁶. Host upregulation of PCSK9 may be a protective immunological response as it prevents entry of the parasite by blocking the recycling of lipid receptors, resulting in impaired clearance of LDL.

We fully acknowledge that mechanistic evidence linking PCSK9 levels to specific host immunologic functions remains to be elucidated.

PCSK9-lipid paradox

Researchers describe patients with HIV/HCV co-infection that show a contradictory increased level of PCSK9 that is associated with reduced plasma LDL and elevated VLDL ²⁹. Elevated PCSK9 levels coupled with reduced LDL levels is articulated as the “PCSK9-lipid paradox” ²⁹ because, typically, high PCSK9 should lead to elevated plasma LDL levels. One hypothesis to explain this observation is that viral particle uptake, and subsequent virion production, requires an increase in cellular cholesterol uptake ^29,30. Thus, hepatocytes reduce LDL uptake by increasing PCSK9 expression. The finding that HIV/HCV infection is also associated with increases in VLDL levels suggests that lipase-mediated VLDL lipolysis is also decreased upon infection ³⁷. This might explain why LDL levels go down—not up— when PCSK9 is increased. While our understanding is still incomplete, these data suggest that there is coordination between host immunity and lipoprotein metabolism ³⁸.

The upregulation of PCSK9 is likely a beneficial response, and this response would be lost amid the liver-specific editing of the PCSK9 locus in individuals with wild-type PCSK9 alleles.

Conclusion: a call to action

Considering the cost and inconvenience of current therapies, an attractive thought is a single administration of a gene editing based therapy to “cure cardiovascular disease” ³⁹. There remains a strong relationship between the incidence of dyslipidemia and the consumption of the Western Diet ^23,40. In the United States, federal food commodity subsidies promote food choices that contribute to diet-related cardiometabolic disease ^41,42. The proposed PCSK9 gene editing intervention is a risky solution, whereas eliminating agricultural subsidies for crops essential for highly processed foods (e.g., high fructose corn syrup derived from corn) seems a safer approach than the genetic modification of humans carrying wild-type PCSK9 alleles. While we, and many others, would consider supporting a permanent gene editing treatment for people where alternative biologics do not exist, this is not the situation for PCSK9. Moreover, providing a PCSK9 gene-editing solution for the treatment of cardiovascular disease assumes PCSK9 only has one function. Here, we provide a summary of some of the evidence for other functions for PCSK9 that have been overlooked by proponents of population-wide editing of wild-type PCSK9. The human genome has only about twenty thousand protein coding genes ⁴³, and rarely does one gene have only one function. Therefore, before inactivating a gene, there must be a thorough understanding of the factors, or selective pressures (biological functions), that have led to the gene’s selection and maintenance in the vertebrate genome. As a principle of genetics, there must be selection at the gene locus for the gene to be retained for millions of years. Recent discussion of this gene editing approach fail to discuss any potential roles of PCSK9 in immunological responses and during pregnancy ^44,45. Without such knowledge, it is premature to utilize gene editing to produce a lifelong inactivation of liver PCSK9 in people expressing wild-type variants.

Highlights.

• A new therapeutic approach aims to treat patients with Familial Hypercholesterolemia (FH) by base-editing the PCSK9 gene and to then expand treatment as a preventative measure to the general population who may be at risk for developing cardiovascular disease.

• The evidence that PCSK9 is implicated in other functions— such as infection, immunity, and pregnancy— may explain why PCSK9 has been maintained in the vertebrate genome for millions of years of evolution.

• There is currently an incomplete understanding of the role of PCSK9 in immunity, and, therefore, inactivating wild-type PCSK9 in otherwise healthy humans as a preventative measure is ethically questionable.

Non-standard abbreviations and acronyms

PCSK9

proprotein convertase subtilisin/Kexin type 9

CFTR

cystic fibrosis transmembrane conductance regulator

FH

familial hypercholesterolemia

HCV

hepatitis C virus

HDL

high density lipoprotein

LDL

low-density lipoprotein

VLDL

very low-density lipoprotein