Predicting humoral responses to primary and booster SARS-CoV-2 mRNA vaccination in people living with HIV: a machine learning approach.

BACKGROUND: SARS-CoV-2 mRNA vaccines are highly immunogenic in people living with HIV (PLWH) on effective antiretroviral therapy (ART). However, whether viro-immunologic parameters or other factors affect immune responses to vaccination is debated. This study aimed to develop a machine learning-based model able to predict the humoral response to mRNA vaccines in PLWH and to assess the impact of demographic and clinical variables on antibody production over time. METHODS: Different machine learning algorithms have been compared in the setting of a longitudinal observational study involving 497 PLWH, after primary and booster SARS-CoV-2 mRNA vaccination. Both Generalized Linear Models and non-linear Models (Tree Regression and Random Forest) were trained and tested. RESULTS: Non-linear algorithms showed better ability to predict vaccine-elicited humoral responses. The best-performing Random Forest model identified a few variables as more influential, within 39 clinical, demographic, and immunological factors. In particular, previous SARS-CoV-2 infection, BMI, CD4 T-cell count and CD4/CD8 ratio were positively associated with the primary cycle immunogenicity, yet their predictive value diminished with the administration of booster doses. CONCLUSIONS: In the present work we have built a non-linear Random Forest model capable of accurately predicting humoral responses to SARS-CoV-2 mRNA vaccination, and identifying relevant factors that influence the vaccine response in PLWH. In clinical contexts, the application of this model provides promising opportunities for predicting individual vaccine responses, thus facilitating the development of vaccination strategies tailored for PLWH.

Spike-Specific Memory B Cell Response in Hematopoietic Cell Transplantation Recipients following Multiple mRNA-1273 Vaccinations: A Longitudinal Observational Study.

Preventing SARS-CoV-2 infection is of utmost importance in allogeneic hematopoietic cell transplantation patients (allo-HCT), given their heightened susceptibility to adverse outcomes associated with SARS-CoV-2 infection. However, limited data are available regarding the immune response to COVID-19 vaccines in these subjects, particularly concerning the generation and persistence of spike-specific memory response. Here, we analyzed the spike-specific memory B cells in a cohort of allo-HCT recipients vaccinated with multiple doses of the mRNA-1273 vaccine and monitored the spike-specific antibody response from baseline up to one month after the fourth dose. After the primary vaccine series, the frequency of spike-specific B cells, detected within the pool of Ig-switched CD19+ cells, significantly increased. The booster dose further induced a significant expansion, reaching up to 0.28% of spike-specific B cells. The kinetics of this expansion were slower in the allo-HCT recipients compared to healthy controls. Spike-specific IgG and ACE2/RBD binding inhibition activity were observed in 80% of the allo-HCT recipients after the first two doses, with a significant increase after the third and fourth booster doses, including in the subjects who did not respond to the primary vaccine series. Additionally, 87% of the allo-HCT recipients exhibited positive cross-inhibition activity against the BA.1 variant. Our findings provide evidence that allo-HCT recipients need repeated doses of the mRNA-1273 vaccine to induceSARS-CoV-2 specific immune response similar to that observed in healthy individuals. This is particularly crucial for vulnerable individuals who may exhibit a limited response to the primary series of SARS-CoV-2 vaccination.

Homologous or heterologous administration of mRNA or adenovirus-vectored vaccines show comparable immunogenicity and effectiveness against the SARS-CoV-2 Omicron variant.

BACKGROUND: Heterologous prime-boost schedules have been employed in SARS-CoV-2 vaccination, yet additional data on immunogenicity and effectiveness are still needed. RESEARCH DESIGN AND METHODS: Here, we measured the immunogenicity and effectiveness in the real-world setting of the mRNA booster dose in 181 subjects who had completed primary vaccination with ChAdOx1, BNT162b2, or mRNA1273 vaccines (IMMUNO_COV study; protocol code 18,869). The spike-specific antibody and B cell responses were analyzed up to 6 months after boosting. RESULTS: After an initial slower antibody response, the heterologous ChAdOx1/mRNA prime-boost formulation elicited spike-specific IgG titers comparable to homologous approaches, while spike-specific B cells showed a higher percentage of CD21-CD27- atypical cells compared to homologous mRNA vaccination. Mixed combinations of BNT162b2 and mRNA-1273 elicited an immune response comparable with homologous strategies. Non-significant differences in the Relative Risk of infection, calculated over a period of 18 months after boosting, were reported among homologous or heterologous vaccination groups, indicating a comparable relative vaccine effectiveness. CONCLUSIONS: Our data endorse the heterologous booster vaccination with mRNA as a valuable alternative to homologous schedules. This approach can serve as a solution in instances of formulation shortages and contribute to enhancing vaccine strategies for potential epidemics or pandemics.

Does Nirmatrelvir/Ritonavir Influence the Immune Response against SARS-CoV-2, Independently from Rebound?

Recurrence of coronavirus disease 19 (COVID-19) symptoms and SARS-CoV-2 viral load relapse have been reported in people treated with nirmatrelvir/ritonavir (NM/r). However, little is understood about the etiology of this phenomenon. Our aim was to investigate the relation between the host's immune response and viral rebound. We described three cases of COVID-19 rebound that occurred after treatment with nirmatrelvir/ritonavir (group A). In addition, we compared spike-specific antibody response and plasma cytokine/chemokine patterns of the rebound cases with those of (i) control patients treated with nirmatrelvir/ritonavir who did not show rebound (group B), and (ii) subjects not treated with any anti-SARS-CoV-2 drug (group C). The anti-spike antibodies and plasma cytokines/chemokines were similar in groups A and B. However, we observed a higher anti-BA.2 spike IgG response in patients without antiviral treatment (group C) [geometric mean titer 210,807, 5.1- and 8.2-fold higher compared to group A (p = 0.039) and group B (p = 0.032)]. Moreover, the patients receiving antiviral treatment (groups A-B) showed higher circulating levels of platelet-derived growth factor subunit B (PDGF-BB) and vascular endothelial growth Factors (VEGF) and lower levels of interleukin-9 (IL-9), interleukine-1 receptor antagonist (IL-1 RA), and regulated upon activation normal T cell expressed and presumably secreted chemokine (RANTES) when compared to group C. In conclusion, we observed lower anti-spike IgG levels and different cytokine patterns in nirmatrelvir/ritonavir-treated patients compared to those not treated with anti-SARS-CoV-2 drugs. This suggests that early antiviral treatment, by reducing viral load and antigen presentation, could mitigate the immune response against SARS-CoV-2. The clinical relevance of such observation should be further investigated in larger populations.

Trajectory of Spike-Specific B Cells Elicited by Two Doses of BNT162b2 mRNA Vaccine.

The mRNA vaccines for SARS-CoV-2 have demonstrated efficacy and immunogenicity in the real-world setting. However, most of the research on vaccine immunogenicity has been centered on characterizing the antibody response, with limited exploration into the persistence of spike-specific memory B cells. Here we monitored the durability of the memory B cell response up to 9 months post-vaccination, and characterized the trajectory of spike-specific B cell phenotypes in healthy individuals who received two doses of the BNT162b2 vaccine. To profile the spike-specific B cell response, we applied the tSNE and Cytotree automated approaches. Spike-specific IgA+ and IgG+ plasmablasts and IgA+ activated cells were observed 7 days after the second dose and disappeared 3 months later, while subsets of spike-specific IgG+ resting memory B cells became predominant 9 months after vaccination, and they were capable of differentiating into spike-specific IgG secreting cells when restimulated in vitro. Other subsets of spike-specific B cells, such as IgM+ or unswitched IgM+IgD+ or IgG+ double negative/atypical cells, were also elicited by the BNT162b2 vaccine and persisted up to month 9. The analysis of circulating spike-specific IgG, IgA, and IgM was in line with the plasmablasts observed. The longitudinal analysis of the antigen-specific B cell response elicited by mRNA-based vaccines provides valuable insights into our understanding of the immunogenicity of this novel vaccine platform destined for future widespread use, and it can help in guiding future decisions and vaccination schedules.

B cell response after SARS-CoV-2 mRNA vaccination in people living with HIV.

BACKGROUND: Limited longitudinal data are available on immune response to mRNA SARS-CoV-2 vaccination in people living with HIV (PLWHIV); therefore, new evidence on induction and persistence of spike-specific antibodies and B cells is needed. METHODS: In this pilot study we investigated the spike-specific humoral and B cell responses up to six months after vaccination with two doses of mRNA vaccines in 84 PLWHIV under antiretroviral therapy compared to 79 healthy controls (HCs). RESULTS: Spike-specific IgG persisted six months in PLWHIV with no significant differences compared to HCs, even though a significantly lower IgG response was observed in patients with CD4+ T cells < 350/mmc. The frequency of subjects with antibodies capable of inhibiting ACE2/RBD binding was comparable between PLWHIV and HCs a month after the second vaccine dose, then a higher drop was observed in PLWHIV. A comparable percentage of spike-specific memory B cells was observed at month six in PLWHIV and HCs. However, PLWHIV showed a higher frequency of spike-specific IgD- CD27- double-negative memory B cells and a significantly lower rate of IgD- CD27+ Ig-switched memory B cells compared to HCs, suggesting a reduced functionality of the antigen-specific memory B population. CONCLUSIONS: The mRNA vaccination against SARS-CoV-2 elicits humoral and B cell responses quantitatively similar between PLWHIV and HCs, but there are important differences in terms of antibody functionality and phenotypes of memory B cells, reinforcing the notion that tailored vaccination policies should be considered for these patients.

SARS-CoV-2 vaccination has been demonstrated to protect people from severe COVID-19 and death. This is achieved through the induction of a specific immune response that recognizes and responds to the virus. Limited data are available on the immune response to SARS-CoV-2 vaccination in people living with HIV (PLWHIV). In this study, we evaluated the immune response up to six months after vaccination with two doses of vaccines in PLWHIV being treated with the standard antiretroviral therapy. We show that the immune response observed in PLWHIV is broadly similar to that in healthy subjects but that there are some differences in the cells induced as part of the immune response. We therefore suggest that specific vaccination policies should be considered for these PLWHIV.

Human neutralizing antibodies to cold linear epitopes and subdomain 1 of the SARS-CoV-2 spike glycoprotein.

Emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike glycoprotein that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera, including the nine human coronaviruses, through recognition of a conserved motif that includes the S2' site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization, and, similar to fp.006 and hr2.016, protects mice expressing human angiotensin-converting enzyme 2 against infection when present as a bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae, including SARS-CoV-2 variants.

Human neutralizing antibodies to cold linear epitopes and to subdomain 1 of SARS-CoV-2.

Emergence of SARS-CoV-2 variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera , including the nine human coronaviruses, through recognition of a conserved motif that includes the S2' site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization and, like fp.006 and hr2.016, protects mice when present as bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae , including SARS-CoV-2 variants. One sentence summary: Broadly cross-reactive antibodies that protect from SARS-CoV-2 variants are revealed by virus coldspot-driven discovery.

Profiling the B cell immune response elicited by vaccination against the respiratory virus SARS-CoV-2.

B cells play a fundamental role in host defenses against viral infections. Profiling the B cell response elicited by SARS-CoV-2 vaccination, including the generation and persistence of antigen-specific memory B cells, is essential for improving the knowledge of vaccine immune responsiveness, beyond the antibody response. mRNA-based vaccines have shown to induce a robust class-switched memory B cell response that persists overtime and is boosted by further vaccine administration, suggesting that memory B cells are critical in driving a recall response upon re-exposure to SARS-CoV-2 antigens. Here, we focus on the role of the B cell response in the context of SARS-CoV-2 vaccination, offering an overview of the different technologies that can be used to identify spike-specific B cells, characterize their phenotype using machine learning approaches, measure their capacity to reactivate following antigen encounter, and tracking the maturation of the B cell receptor antigenic affinity.

The third dose of mRNA SARS-CoV-2 vaccines enhances the spike-specific antibody and memory B cell response in myelofibrosis patients.

Vaccination against SARS-CoV-2 using mRNA-based vaccines has been highly recommended for fragile subjects, including myelofibrosis patients (MF). Available data on the immune responsiveness of MF patients to mRNA SARS-CoV-2 vaccination, and the impact of the therapy with the JAK inhibitor ruxolitinib, are still fragmented. Here, we profile the spike-specific IgG and memory B-cell response in MF patients, treated or not with ruxolitinib, after the second and the third dose of SARS-CoV-2 BNT162b2 (BioNTech) and mRNA-1273 (Moderna) vaccines. Plasma and peripheral blood mononuclear cells samples were collected before vaccination, post the second and the third doses and tested for spike-specific antibodies, ACE2/RBD antibody inhibition binding activity and spike-specific B cells. The third vaccine dose significantly increased the spike-specific IgG titers in both ruxolitinib-treated and untreated patients, and strongly enhanced the percentage of subjects with antibodies capable of in vitro blocking ACE2/RBD interaction, from 50% up to 80%. While a very low frequency of spike-specific B cells was measured in blood 7 days after the second vaccination dose, a strong and significant increase was elicited by the third dose administration, generating a B cell response similar to the one detected in healthy controls. Despite the overall positive impact of the third dose in MF patients, two patients that were under active concomitant immunosuppressive treatment at the time of vaccination, and a patient that received lymphodepleting therapies in the past, remained low responders. The third mRNA vaccine dose strongly increases the SARS-CoV-2 specific humoral and B cell responses in MF patients, promoting a reactivation of the immune response similar to the one observed in healthy controls.

Monitoring Anti-PEG Antibodies Level upon Repeated Lipid Nanoparticle-Based COVID-19 Vaccine Administration.

PEGylated lipids are one of the four constituents of lipid nanoparticle mRNA COVID-19 vaccines. Therefore, various concerns have been raised on the generation of anti-PEG antibodies and their potential role in inducing hypersensitivity reactions following vaccination or in reducing vaccine efficacy due to anti-carrier immunity. Here, we assess the prevalence of anti-PEG antibodies, in a cohort of vaccinated individuals, and give an overview of their time evolution after repeated vaccine administrations. Results indicate that, in our cohort, the presence of PEG in the formulation did not influence the level of anti-Spike antibodies generated upon vaccination and was not related to any reported, serious adverse effects. The time-course analysis of anti-PEG IgG showed no significant booster effect after each dose, whereas for IgM a significant increase in antibody levels was detected after the first and third dose. Data suggest that the presence of PEG in the formulation does not affect safety or efficacy of lipid-nanoparticle-based COVID-19 vaccines.

The Slower Antibody Response in Myelofibrosis Patients after Two Doses of mRNA SARS-CoV-2 Vaccine Calls for a Third Dose.

Immunization with mRNA SARS-CoV-2 vaccines has been highly recommended and prioritized in fragile subjects, including patients with myelofibrosis (MF). Available data on the vaccine immune response developed by MF patients and the impact of ruxolitinib treatment are still too fragmented to support an informed decision on a third dose for this category of subjects. Here, we show that 76% of MF patients develop spike-specific IgG after the second mRNA SARS-CoV-2 vaccine dose, but the response has a slower kinetics compared to healthy subjects, suggesting a reduced capability of their immune system to promptly react to vaccination. A reduced ACE2/RBD binding inhibition activity of spike-specific antibodies was also observed, especially in ruxolitinib-treated patients. Our results, showing slow kinetics of antibody responses in MF patients following vaccination with mRNA SARS-CoV-2 vaccines, support the need for a third vaccine dose.

Evidence of SARS-CoV-2-Specific Memory B Cells Six Months After Vaccination With the BNT162b2 mRNA Vaccine.

SARS-CoV-2 mRNA vaccines have demonstrated high efficacy and immunogenicity, but limited information is currently available on memory B cell generation and long-term persistence. Here, we investigated spike-specific memory B cells and humoral responses in 145 subjects, up to 6 months after the BNT162b2 vaccine (Comirnaty) administration. Spike-specific antibodies peaked 7 days after the second dose and significant antibody titers and ACE2/RBD binding inhibiting activity were still observed after 6 months, despite a progressive decline over time. Concomitant to antibody reduction, spike-specific memory B cells, mostly IgG class-switched, increased in the blood of vaccinees and persisted 6 months after vaccination. Following the in vitro restimulation, circulating memory B cells reactivated and produced spike-specific antibodies. A high frequency of spike-specific IgG+ plasmablasts, identified by computational analysis 7 days after boost, positively correlated with the generation of IgG+ memory B cells at 6 months. These data demonstrate that mRNA BNT162b2 vaccine elicits strong B cell immunity with spike-specific memory B cells that still persist 6 months after vaccination, playing a crucial role for a rapid response to SARS-CoV-2 virus encounter.

Long-Term Anti-Bacterial Immunity against Systemic Infection by Salmonella enterica Serovar Typhimurium Elicited by a GMMA-Based Vaccine.

Salmonella Typhimurium (STm) represents the most prevalent cause of invasive non-typhoidal Salmonella (iNTS) disease, and currently no licensed vaccine is available. In this work we characterized the long-term anti-bacterial immunity elicited by a STm vaccine based on Generalized Modules of Membrane Antigens (GMMA) delivering O:4,5 antigen, using a murine model of systemic infection. Subcutaneous immunization of mice with STmGMMA/Alhydrogel elicited rapid, high, and persistent antigen-specific serum IgG and IgM responses. The serum was bactericidal in vitro. O:4,5-specific IgG were also detected in fecal samples after immunization and positively correlated with IgG observed in intestinal washes. Long-lived plasma cells and O:4,5-specific memory B cells were detected in spleen and bone marrow. After systemic STm challenge, a significant reduction of bacterial load in blood, spleen, and liver, as well as a reduction of circulating neutrophils and G-CSF glycoprotein was observed in STmGMMA/Alhydrogel immunized mice compared to untreated animals. Taken together, these data support the development of a GMMA-based vaccine for prevention of iNTS disease.

Short or Long Interval between Priming and Boosting: Does It Impact on the Vaccine Immunogenicity?

Characterizing the impact of the vaccination schedule on the induction of B and T cell immune responses is critical for improving vaccine immunogenicity. Here we compare the effect of a short (4 weeks) or a long (18 weeks) interval between priming and boosting in mice, using a model vaccine formulation based on the chimeric tuberculosis vaccine antigen H56 combined with alum. While no significant difference was observed in serum antigen-specific IgG response and the induction of antigen-specific T follicular helper cells into draining lymph nodes after the two immunization schedules, a longer interval between priming and boosting elicited a higher number of germinal center-B cells and H56-specific antibody-secreting cells and modulated the effector function of reactivated CD4+ T cells. These data show that the scheduling of the booster immunization could affect the immune response elicited by vaccination modulating and improving the immunogenicity of the vaccine.

Maraviroc as a potential HIV-1 latency-reversing agent in cell line models and ex vivo CD4 T cells.

Recent studies have suggested that the CCR5 antagonist maraviroc (MVC) may exert an HIV-1 latency reversal effect. This study aimed at defining MVC-mediated induction of HIV-1 in three cell line latency models and in ex vivo CD4 T cells from six patients with suppressed viraemia. HIV-1 induction was evaluated in TZM-bl cells by measuring HIV-1 LTR-driven luciferase expression, and in ACH-2 and U1 latently infected cell lines by measuring cell-free (CFR) and cell-associated (CAR) HIV-1 RNA by qPCR. NF-κB p65 was quantified in nuclear extracts by immunodetection. In ex vivo CD4 T cells, CAR, CFR and cell-associated DNA (CAD) were quantified at baseline and 1-7-14 days post-induction (T1, T7, T14). At T7 and T14, the infectivity of the CD4 T cells co-cultured with MOLT-4/CCR5 target cells was evaluated in the TZM-bl assay (TZA). Results were expressed as fold activation (FA) with respect to untreated cells. No LTR activation was observed in TZM-bl cells at any MVC concentration. NF-κB activation was only modestly upregulated (1.6±0.4) in TZM-bl cells with 5 µM MVC. Significant FA of HIV-1 expression was only detected at 80 µM MVC, namely on HIV-1 CFR in U1 (3.1±0.9; P=0.034) and ACH-2 cells (3.9±1.4; P=0.037). CFR was only weakly stimulated at 20 µM in ACH-2 (1.7±1.0 FA) cells and at 5 µM in U1 cells (1.9±0.5 FA). Although no consistent pattern of MVC-mediated activation was observed in ex vivo experiments, substantial FA values were detected sparsely on individual samples with different parameters. Notably, in one sample, MVC stimulated all parameters at T7 (2.3±0.2 CAD, 6.8±3.7 CAR, 18.7±16.7 CFR, 7.3±0.2 TZA). In conclusion, MVC variably induces HIV-1 production in some cell line models not previously used to test its latency reversal potential. In ex vivo CD4 T cells, MVC may exert patient-specific HIV-1 induction; however, clinically relevant patterns, if any, remain to be defined.

Shelter from the cytokine storm: pitfalls and prospects in the development of SARS-CoV-2 vaccines for an elderly population.

The SARS-CoV-2 pandemic urgently calls for the development of effective preventive tools. COVID-19 hits greatly the elder and more fragile fraction of the population boosting the evergreen issue of the vaccination of older people. The development of a vaccine against SARS-CoV-2 tailored for the elderly population faces the challenge of the poor immune responsiveness of the older population due to immunosenescence, comorbidities, and pharmacological treatments. Moreover, it is likely that the inflammaging phenotype associated with age could both influence vaccination efficacy and exacerbate the risk of COVID-19-related "cytokine storm syndrome" with an overlap between the factors which impact vaccination effectiveness and those that boost virulence and worsen the prognosis of SARS-CoV-2 infection. The complex and still unclear immunopathological mechanisms of SARS-CoV-2 infection, together with the progressive age-related decline of immune responses, and the lack of clear correlates of protection, make the design of vaccination strategies for older people extremely challenging. In the ongoing effort in vaccine development, different SARS-CoV-2 vaccine candidates have been developed, tested in pre-clinical and clinical studies and are undergoing clinical testing, but only a small fraction of these are currently being tested in the older fraction of the population. Recent advances in systems biology integrating clinical, immunologic, and omics data can help to identify stable and robust markers of vaccine response and move towards a better understanding of SARS-CoV-2 vaccine responses in the elderly.

High rate of HDR in gene editing of p.(Thr158Met) MECP2 mutational hotspot.

Rett syndrome is a progressive neurodevelopmental disorder which affects almost exclusively girls, caused by variants in MECP2 gene. Effective therapies for this devastating disorder are not yet available and the need for tight regulation of MECP2 expression for brain to properly function makes gene replacement therapy risky. For this reason, gene editing with CRISPR/Cas9 technology appears as a preferable option for the development of new therapies. To study the disease, we developed and characterized a human neuronal model obtained by genetic reprogramming of patient-derived primary fibroblasts into induced Pluripotent Stem Cells. This cellular model represents an important source for our studies, aiming to correct MECP2 variants in neurons which represent the primarily affected cell type. We engineered a gene editing toolkit composed by a two-plasmid system to correct a hotspot missense variant in MECP2, c.473 C > T (p.(Thr158Met)). The first construct expresses the variant-specific sgRNA and the Donor DNA along with a fluorescent reporter system. The second construct brings Cas9 and targets for auto-cleaving, to avoid long-term Cas9 expression. NGS analysis on sorted cells from four independent patients demonstrated an exceptionally high editing efficiency, with up to 80% of HDR and less than 1% of indels in all patients, outlining the relevant potentiality of the approach for Rett syndrome therapy.