Extensive adaptive immune response of AAVs and Cas proteins in non-human primates

The CRISPR-mediated Cas system is the most widely used tool in gene editing and gene therapy for its convenience and efficiency. Delivery of the CRISPR system by adeno-associated viruses (AAVs) is currently the most promising approach to gene therapy. However, pre-existing adaptive immune responses against CRISPR nuclease (PAIR-C) and AAVs has been found in human serum, indicating that immune response is a problem that cannot be ignored, especially for in vivo gene correction. Non-human primates (NHPs) share many genetic and physiological traits with human, and are considered as the bridge for translational medicine. However, whether NHPs have same PAIR-C status with human is still unknown. Here, macaques (rhesus and cynomolgus), including normal housed and CRISPR-SpCas9 or TALENs edited individuals, were used to detect PAIR-C which covered SaCas9, SpCas9, AsCas12a and LbCas12a. Dogs and mice were also detected to expand the range of species. In addition, pre-existing adaptive antibodies to AAV8 and AAV9 were performed against macaques of different ages. The results showed that adaptive immunity was pre-existing in the macaques regardless of Cas proteins and AAVs. These findings indicate that the pre-existing adaptive immune of AAV-delivered CRISPR construction and correction system should be concerned for in vivo experiments.

Introduction 49 application. 50 The CRISPR system is mainly derived from bacteria and archaea [25], which often 51 invade organisms as pathogens, and are recognized as an alien by the immune system 52 of these organisms, activates lymphocytes in the body to produce corresponding 53 effector cells, and removes them [26]. It is difficult for humans or animals to avoid 54 contact with microorganisms under normal living conditions, and antibodies specific to 55 these pathogens are usually present in the body. Several research groups have found the 56 pre-existing adaptive immune responses against CRISPRCas9 nuclease (PAIR-C) are 57 extensively existing in human, which focus on specific humoral and cellular immunity 58 against SaCas9 and SpCas9[19,20]. These findings raise concerns on the safety of the 59 in vivo CRISPR editing system. Therefore, systematic preclinical assessment of PAIR-60 C using proper laboratory animals is necessary.

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AAVs have been widely used as a vector for gene therapy in preclinical and 62 clinical trials because it is not pathogenic. However, human and Non-human primates 63 (NHPs), as natural hosts for AAVs, have been generally found to neutralize antibodies 64 (NABs) against AAVs, which affect transduction efficiency in vivo [27,28]. Studies 65 have shown that the capsid protein of AAVs can also be recognized by T cells, and 66 cellular immunity of AAVs cannot be ignored [29,30].

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116 The macaque serum PAIR-C is extensively existed regardless of gene editing

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In order to detect antibodies against Cas proteins in macaques, we selected 24 118 healthy wild-type experimental macaques to collect serum and divided into four groups 119 according to age: under 1 year old (n = 3), 1-3 years old (n = 8), 4-8 years old (n = 7) 120 and over 9 years old (n = 6). At the same time, 11 CRISPR-SpCas9 edited macaques (6 121 positive and 5 negative) and 10 TALENs edited macaques (5 positive and 5 negative) 122 were collected for further analysis (S1 Table). The four proteins of SaCas9, SpCas9, 123 AsCas12a, and LbCas12a were purified and subjected to detect PAIR-C level in above 124 monkeys through western blot. The diluted sera were used as a primary antibody to 125 bind Cas proteins, and anti-monkey IgG H&R was used as a secondary antibody to 126 detect IgG capable of binding to Cas proteins. The antibody was detected by automatic 127 exposure to protein chemiluminescence imager. We found that except for the three 128 macaques under the age of one, most of monkey serum samples contain antibodies to 129 the four Cas proteins (S1 Data, S4 To determine and compare the status and levels of PAIR-C in monkeys, we 137 selected 10 dogs (S2 Table) and 16 SPF mice for comparison (S3 Table). Based on the 138 results of western blot, we found that the presence of adaptive antibodies to these four 139 proteins is also prevalent in dog serum as in monkey (Data S1). Unlike previous 140 predictions, antibodies to these four nucleases were also detected in SPF mice (Data 5 141 S1). Statistical results indicate that all dog serum samples were present for antibodies 142 to these four Cas proteins (Table 1, S5 Table). Moreover, 15 of the 16 SPF mice 143 detected antibodies against these four Cas proteins ( 155 According to ELISA results, we found that antibody levels in macaques tend to increase 156 with age and remain relatively stable after adulthood (Fig 2a). One-way analysis of 157 variance (ANOVA) of wild-type monkeys, dogs, and mice showed that there were 158 significant differences between monkeys and mice, as well as between dogs and mice.
159 But there was no significant difference between monkeys and dogs (Fig 2b). We also 160 performed a one-way ANOVA of the macaque positive and negative groups edited by 161 CRISPR-Cas9 and TALENs, with no significant differences (Fig 2c and 2d). At the 162 same time, the wild-type population was compared with the monkey population edited 163 by CRISPR-Cas9 and TALENs, and no significant difference was found (Fig 2e).