Pre-existing immune response to bacterial Cas9 proteins in people with cystic fibrosis

Anti-coagulated peripheral blood was collected by venous puncture and plasma samples collected following centrifugation at 16,000×for 10 min at 4in MicrotainerBlood Collection Tubes (BD catalog no. 365967). Blood from non-CF and people with CF was provided as de-identified biospecimens from the Cure CF Columbus Research Development Program Translational and Data Core under the NCH IRB and OSU IRB 2020H0399. Several additional non-CF samples were provided from OSU IRB 2014H0154. Plasma samples were stored at -80until use for ELISA endpoint measurements. PBMC were isolated by gradient centrifugation utilizing Ficoll-Paque Plus (Sigma, catalog no. GE17-1440-02) according to manufacturer's instructions. Here, 1 × 10total cells were resuspended in 90% HI-FBS + 10% DMSO and cryopreserved for storage in liquid nitrogen. g ° C ° C ® 7

Samples were processed in batches, where n = 11 samples were thawed and used at the same time for assessment in four endpoint ELISAs for human serum albumin (HSA), tetanus toxoid,Cas9, orCas9. ELISA methods were adapted from those previously reported. Cas9 (Sa Cas9) SaCas9 without a nuclear localization signal (No NLS) andCas9 (Sp Cas9) SpCas9 V3 No NLS were obtained from IDT. HSA was obtained from Sigma Aldrich (catalog no. SRP6182) and inactivated tetanus toxoid was obtained from Cellero (catalog no. 1002). Nunc Maxisorp ELISA plates (Fisher catalog no. 12-565-135) were coated overnight at 4by dilution into 100 l of carbonate-bicarbonate coating buffer (Sigma Aldrich, catalog no. C3041-50CAP) to 0.5 g of antigen per well for each of the recombinant Cas9 proteins, tetanus toxoid, or HSA. Following overnight coating, wells were washed 3 times with Tris buffered saline with Tween-20 (Sigma Aldrich, catalog no. T9039-10PK), and plates were incubated with 200 l blocking solution for 1 hour at room temperature. Blocker BSA (10%) from ThermoFisher (catalog no. 37525) was diluted to 1% in phosphate buffered saline (PBS) for the blocking solution. Sample dilutions were performed in blocking solution (1% BSA in PBS) and were plated in technical duplicates. Plates were washed three times following blocking and 100 l of samples were added to each well and incubated at room temperature for 2 hours. Plates were washed 3 times, followed by incubation with 200 l of diluted secondary antibody for 1 hr at room temperature. The goat polyclonal, goat anti-human IgG Fc secondary antibody (R&D Systems, catalog no. NB7449) was diluted 1:100,000 in 1% BSA. Plates were washed an additional three times and 100 l of room temperature development reagent was added to each well. Plates were incubated at room temperature protected from light; a timer was used to precisely track the development time of each plate, and 50 l of 1 N sulfuric acid was added after 10 min to end the reactions. The absorbance at 450 nm was quantified using a Spectramax ID3 plate reader. S. aureus S. pyogenes S. aureus S. pyogenes ⁰⁴¹ ° C μ μ μ μ μ μ μ

Peptide stimulations were performed as previously described.Frozen PBMC were rapid thawed in complete RPMI containing 2 l benzonase (Sigma, catalog no. 706643), centrifuged at 350×for 10 min at 4 °C, and resuspended in a 96-well U-bottom plate (5 × 10cells/well). Media, definedorpeptide pools (), or Peptivator CEF MHC Class I Plus positive control peptide pool (Miltenyi, catalog no. 130-098-426) was added to cells with brefeldin A (eBioscience, catalog no. 00-4506-51), recombinant human interleukin (IL)-2 (Sigma, catalog no. 11011456001) and anti-human CD107a AF700 (BD, catalog no. 561340) were added to wells and incubated at 37 °C for 24 hr. Cell stimulation cocktail containing PMA/I (Invitrogen, catalog no. 00-4970) was added to unstimulated wells for the final 6 hr of the incubation period. Following incubation, cells were labeled with Live/Dead Fixable Blue Dead Cell Stain Kit (Invitrogen, catalog no. L34962) according to manufacturer instructions. Samples were then blocked with Human TrueStain FcX (BioLegend, catalog no. 422302) for 10 min at room temperature and subsequently surface stained with the following fluorescently conjugated antibodies for 30 min on ice: anti-human CD3 APC-H7 (BD, catalog no. 560176), anti-human CD4 BUV496 (BD, catalog no. 612936, anti-human CD8 BUV805 (BD, catalog no. 612889), anti-human CD45R0 BV570 (BioLegend, catalog no. 304226), anti-human CD45RA AF647 (BioLegend, catalog no. 305154). Samples were fixed and permeabilized with BioLegend True-Nuclear Transcription Factor Buffer Kit (catalog no. 424401) according to manufacturer's instructions. Following permeabilization, samples were blocked as described above and stained with the following antibodies specific for intracellular antigens for 40 min on ice: anti-human FoxP3 Pacific Blue (BioLegend, catalog no. 320116), anti-human IFN-γ BB700 (BD, catalog no. 566394), anti-human TNF BV750 (BD, catalog no. 566359) anti-human granzyme B FITC (BioLegend, catalog no. 515403). Samples were washed and resuspended in PBS to run on a Cytek Aurora flow cytometer. Samples were analyzed using FlowJo (BD). Upstream of representative flow panels and populations shown on graphs samples were gated on viable CD3CD4CD8cells (CD4 T cells) or viable CD3CD8CD4cells (CD8 T cells). ^{041 041 041 041} Table 1 μ g S. aureus S. pyogenes 5 , + + − + + −

Statistical analyses and calculations of the area under the curve were performed in GraphPad Prism Version 10.4.1. The specific statistical tests used are listed in individual figure legends. GraphPad Prism Version 10.4.1 was used for all statistical analysis.

To evaluate whether antibodies to recombinant Cas9 proteins exist in people with CF, an ELISA-based method was adapted for this study.Samples were obtained from non-CF healthy control and people with CF. We obtained 24 non-CF control samples from individuals that were 79% female with a median age of 32.5 yr. Thirty-five samples were collected from individuals with CF that were 57% female with a median age of 24 yr. ELISAs were performed to detect relative levels of anti-Cas9 (Sa Cas9) and anti-Cas9 (Sp Cas9) IgG utilizing anti-HSA IgG as a negative control, and anti-tetanus toxoid IgG as a positive control.Based on the dilution curves, IgG levels between non-CF control and CF are similar for each antibody specificity (). Calculation of the area under the curve (AUC) to quantify antigen-specific IgG levels () revealed statistically significant increases in the levels of tetanus toxoid, Sa Cas9, and Sp Cas9 compared to HSA for non-CF control and CF, although there were no significant differences when comparing non-CF control or CF responses to either Cas9 or the tetanus toxoid positive control. ^{041 041} Fig. 1A Fig. 1B S. aureus S. pyogenes

We performed an additional more stringent analysis by assessing whether individual samples had positive antibody responses for Cas9 or tetanus toxoid defined by an AUC greater than 3 standard deviations above the mean AUC for the HSA negative control (from data shown in). As expected, antibody positivity to the positive control tetanus toxoid was high, with 89% of all samples having positive tetanus toxoid antibody responses, with no difference between non-CF control (87%) and CF samples (91%). We detected positive antibody responses to(Sa) Cas9 in 58% of non-CF control and 57% of CF samples, while 52% of non-CF control and 75% of CF samples had positive antibody responses to(Sp) Cas9. Overall, these results suggest that the prevalence of pre-existing antibody responses to Sa Cas9 and Sp Cas9 is relatively high in both non-CF and CF populations, consistent with previous findings reported in a cohort of healthy humans. Fig. 1B ⁰⁴¹ S. aureus S. pyogenes

While antibodies are indicative of pre-existing immunity that may correlate to immune responses targeting Cas9 proteins or Cas9 gene-edited cells, cytotoxic CD8 and CD4 T cell responses could result in the functional targeting of gene-edited cells expressing Cas9 via MHCI and MCHII, respectively. Therefore, we next investigated T cell immunity in a small cohort of samples from non-CF controls (n = 8, median age 39.5 yr, 83% female) and CF (n = 6, median age 31.5 yr, 63% female). PBMC were stimulated with a number of previously identifiedandimmunostimulatory Cas9 peptides, or CEF or PMA and ionomycin (PMA/I) as positive controls to determine if T cells from people with CF have altered responses specific fororCas9 peptides compared to non-CF controls.Following 24 hr of stimulation, flow cytometry analyses were performed to quantify the frequency of naive, memory, and regulatory T cells (Treg). Although T cell immunity in CF has not been widely reported in the literature, FoxP3CD4 Treg have been previously described to be significantly reduced in CF.Specifically, reduced Treg responses in CF with chronicinfection have been observed.While the Treg frequency was significantly increased in vitro following HEMT treatment,whether these levels are rescued to levels equivalent to non-CF control individuals is unknown. Despite the small cohort in our study, we confirmed that the frequency of peripheral Foxp3CD4 T cells was significantly reduced in CF compared to controls (). We also determined that the frequencies of CD45RA(generally naïve) and CD45RO(generally memory) CD4 () and CD8 () T cells in the peripheral blood revealed no dramatic changes between non-CF control and CF samples, suggesting the distribution of naïve and memory T cell populations in the peripheral blood is unaltered in people with CF. S. aureus S. pyogenes S. aureus S. pyogenes P. aeruginosa ^{041 041 041 041 041} Fig. 2A Fig. 2B Fig. 2C + , + + +

To determine whether the cytotoxic ability of CD4 and CD8 T cell responses was altered in CF, we measured the intracellular expression of granzyme B (gzmB) and found no significant differences in the percentage of gzmBCD4 () or CD8 () T cells between non-CF and CF samples. Additionally, we included fluorescently labeled anti-CD107a antibody during a 6 hr stimulation with PMA/I in the presence of IL-2 and brefeldin A to serve as a proxy measure of cytotoxic degranulation, as the antibody would bind CD107a expressed on the surface of cells during degranulation for analysis by flow cytometry.The frequency of CD107aCD4 () or CD8 () T cells was not altered in CF compared to non-CF controls. Together, these findings demonstrate no alteration in cytotoxic molecule expression in CF compared to non-CF control T cells. + + Fig. 3A Fig. 3B ⁰⁴¹ Fig. 3C Fig. 3D

As anticipated based on previous studies identifying the peptides utilized (), we detected low frequencies of CD4 T cells producing IFN-γ and TNF following stimulations (), although responses specific to the(Sa) peptide pool were largely undetectable. However, CD4 IFN-γ/TNF production in response to both(Sp) peptide pools was similar to the CEF positive control peptide pool, and responses were equivalent between control samples and CF (). Similar results were observed when we assessed IFN-γ and TNF production by CD8 T cells following stimulation with Sa, Sp, or CEF peptide pools (). In summary, these results indicate that pre-existing effector CD8 and Th1 CD4 T cell responses specific to Cas9 can be detected and are equivalent between non-CF controls and CF samples. Critically, these finding suggest there may be no additional or enhanced barrier to development of gene therapy treatments in individuals with CF compared to non-CF populations due to similar levels pre-existing Cas9-specific antibody and T cell responses. Table 1 Fig. 3E Fig. 3E Fig. 3F S. aureus S. pyogenes

Although we observed no significant differences in the ability of non-CF control or CF CD4 or CD8 T cells to produce IFN-γ/TNF in response to,, or CEF peptides, we noted a striking and significant reduction in the frequency of both CD4 () and CD8 () CF T cells producing IFN-γ/TNF following stimulation with PMA/I compared to non-CF controls. This suggests that while the antigen-specific responses investigated were equivalent between non-CF control and CF samples, the maximal effector response is specifically limited in CF. Whether these CF T cells responses are blunted in response to infection or health status of the blood donors or other stimuli remains unknown. In summary, while this study indicates no additional risks for adaptive immune-mediated elimination of Cas9 targeted cells that would hamper gene targeting efforts in CF, we have identified a previously unreported defect in the ability of Th1 and CD8 T cells to produce IFN-γ/TNF in response to PMA/I. S. pyogenes S. aureus Fig. 3E Fig. 3F