The potential risks of C-C chemokine receptor 5-edited babies in bone development

Abstract

Hutter et al.1 first reported that a bone marrow transplant using stem cells derived from a donor with homozygous CCR5 delta32 gene mutation remained HIV-positive but virus-free (below the limits of detection) after halting antiretroviral therapy. Since this observation in 2009, mutation of the CCR5 gene has become an important target in the prevention and treatment of HIV infection. The CRISPR-Cas9 system, which has been called the biggest biotech discovery in the history of molecular biology, can be used for precise genome engineering with the aim of treating genetic disorders. Currently, the application of gene-editing tools, such as CRISPR-Cas9, for genetic engineering of embryos for use in assisted reproduction is prohibited in much of Europe, the United States, and China.2,3 However, at the Second International Summit on Human Genome Editing in Hong Kong (http://www.nationalacademies.org/), Jiankui He claimed that his team had used CRISPR-Cas9 systems to successfully edit the CCR5 gene in twin baby girls, Lulu and Nana. In Lulu, one copy of exon 3 in the CCR5 gene has an inserted base, with the other copy missing four bases. In Nana, a 15-nucleotide deletion (delta15) within one copy of CCR5 was described, with the other copy of the CCR5 gene remaining intact. \n \nThe CCR5 gene is located at chromosome region 3p21.314 and comprises three exons, two introns and two promoters.5 The C-C chemokine receptor 5 (CCR5) protein encoded by the CCR5 gene consists of 352 amino acids6 and is composed of a conserved, N-terminal seven trans-membrane domain and a C-terminal tail.7 This structure is important for the binding of HIV glycoprotein receptors to host cells and HIV co-receptor CD4 activity.8 Samson et al. found that the second extracellular loop of CCR5 is specifically affected by delta32 mutations in exon 3, which result in the absence of the final three trans-membrane domains in addition to regions involved in G-protein interaction and signal transduction. In CD4+\u2009cells, this mutation inhibits CCR5 protein expression on the cell surface, thereby preventing HIV envelope fusion.9 Moreover, the presence of the mutant delta32 protein in the endoplasmic reticulum inhibits transport of the wild-type CCR5 protein to the cell surface via a trans-dominant mechanism.10 Because most strains of HIV use CCR5 to enter host cells, the deletion of both copies of the CCR5 gene (not one copy) protects against HIV infection.11,12 Thus, Nana would still be susceptible to HIV infection. Although He demonstrated that Lulu was homozygous for the disrupted CCR5 gene, this child may also be genetically mosaic, which means that Lulu may carry some edited cells and some unedited cells. Furthermore, although He claimed an absence of dangerous off-target mutations in both twins based on single cell sequencing studies, these results were not peer-reviewed and confirmed by an independent team. Therefore, Lulu’s genetic status should be continually monitored throughout her life, and it is possible that she may encounter unpredictable disorders in the future. \n \nRole of CCR5 deficiency in diseases \nIndividuals who are naturally homozygous for the delta32 mutation, which abolishes CCR5 expression, are generally healthy and at no apparent disadvantage.8 However, apart from the protective effects against HIV infection, the impacts of this mutation, positive or negative, on other diseases are open to debate.13 To date, several studies have indicated that CCR5 delta32 mutations provide significant resistance to smallpox,14 in addition to enhancing certain forms of memory15 but also render individuals more vulnerable to influenza16 and the West Nile virus.17 In mice, CCR5 deficiency exacerbates stroke-related brain injury.18 CCR5 is thought to mediate pro-inflammatory effects in the pathogenesis of rheumatoid arthritis (RA).19 However, Fleishaker et al.20 reported that a CCR5 antagonist (maraviroc), which has been approved for use in HIV patients, was ineffective in treating patients with RA who had not responded to methotrexate (MTX). Moreover, a double-blind, placebo-controlled trial in 2015 found that maraviroc was associated with reduced bone loss at the hip and lumbar spine of HIV-infected patients.21 Other studies demonstrated direct roles of CCR5 in osteoclastogenesis and osteoclast-osteoblast communication.22,23 These clinical and basic investigations highlight the skeletal effects associated with the functional loss of CCR5.24