Long-term outcomes after gene therapy for adenosine deaminase severe combined immune deficiency.

Patients lacking functional adenosine deaminase activity have severe combined immunodeficiency (ADA SCID), which can be treated with ADA enzyme replacement therapy (ERT), allogeneic hematopoietic stem cell transplantation (HSCT), or autologous HSCT with gene-corrected cells (gene therapy [GT]). A cohort of 10 ADA SCID patients, aged 3 months to 15 years, underwent GT in a phase 2 clinical trial between 2009 and 2012. Autologous bone marrow CD34+ cells were transduced ex vivo with the MND (myeloproliferative sarcoma virus, negative control region deleted, dl587rev primer binding site)-ADA gammaretroviral vector (gRV) and infused following busulfan reduced-intensity conditioning. These patients were monitored in a long-term follow-up protocol over 8 to 11 years. Nine of 10 patients have sufficient immune reconstitution to protect against serious infections and have not needed to resume ERT or proceed to secondary allogeneic HSCT. ERT was restarted 6 months after GT in the oldest patient who had no evidence of benefit from GT. Four of 9 evaluable patients with the highest gene marking and B-cell numbers remain off immunoglobulin replacement therapy and responded to vaccines. There were broad ranges of responses in normalization of ADA enzyme activity and adenine metabolites in blood cells and levels of cellular and humoral immune reconstitution. Outcomes were generally better in younger patients and those receiving higher doses of gene-marked CD34+ cells. No patient experienced a leukoproliferative event after GT, despite persisting prominent clones with vector integrations adjacent to proto-oncogenes. These long-term findings demonstrate enduring efficacy of GT for ADA SCID but also highlight risks of genotoxicity with gRVs. This trial was registered at www.clinicaltrials.gov as #NCT00794508.

Lentiviral globin gene therapy with reduced-intensity conditioning in adults with ß-thalassemia: a phase 1 trial.

ß-Thalassemias are inherited anemias that are caused by the absent or insufficient production of the ß chain of hemoglobin. Here we report 6-8-year follow-up of four adult patients with transfusion-dependent ß-thalassemia who were infused with autologous CD34+ cells transduced with the TNS9.3.55 lentiviral globin vector after reduced-intensity conditioning (RIC) in a phase 1 clinical trial ( NCT01639690) . Patients were monitored for insertional mutagenesis and the generation of a replication-competent lentivirus (safety and tolerability of the infusion product after RIC-primary endpoint) and engraftment of genetically modified autologous CD34+ cells, expression of the transduced ß-globin gene and post-transplant transfusion requirements (efficacy-secondary endpoint). No unexpected safety issues occurred during conditioning and cell product infusion. Hematopoietic gene marking was very stable but low, reducing transfusion requirements in two patients, albeit not achieving transfusion independence. Our findings suggest that non-myeloablative conditioning can achieve durable stem cell engraftment but underscore a minimum CD34+ cell transduction requirement for effective therapy. Moderate clonal expansions were associated with integrations near cancer-related genes, suggestive of non-erythroid activity of globin vectors in stem/progenitor cells. These correlative findings highlight the necessity of cautiously monitoring patients harboring globin vectors.

SARS-CoV-2 Genomic Variation in Space and Time in Hospitalized Patients in Philadelphia.

The severe acute respiratory coronavirus 2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. The epidemic accelerated in Philadelphia, PA, in the spring of 2020, with the city experiencing a first peak of infections on 15 April, followed by a decline through midsummer. Here, we investigate spread of the epidemic in the first wave in Philadelphia using full-genome sequencing of 52 SARS-CoV-2 samples obtained from 27 hospitalized patients collected between 30 March and 17 July 2020. Sequences most commonly resembled lineages circulating at earlier times in New York, suggesting transmission primarily from this location, though a minority of Philadelphia genomes matched sequences from other sites, suggesting additional introductions. Multiple genomes showed even closer matches to other Philadelphia isolates, suggestive of ongoing transmission within Philadelphia. We found that all of our isolates contained the D614G substitution in the viral spike and belong to lineages variously designated B.1, Nextstrain clade 20A or 20C, and GISAID clade G or GH. There were no viral sequence polymorphisms detectably associated with disease outcome. For some patients, genome sequences were determined longitudinally or concurrently from multiple body sites. In both cases, some comparisons showed reproducible polymorphisms, suggesting initial seeding with multiple variants and/or accumulation of polymorphisms after infection. These results thus provide data on the sources of SARS-CoV-2 infection in Philadelphia and begin to explore the dynamics within hospitalized patients.IMPORTANCE Understanding how SARS-CoV-2 spreads globally and within infected individuals is critical to the development of mitigation strategies. We found that most lineages in Philadelphia had resembled sequences from New York, suggesting infection primarily but not exclusively from this location. Many genomes had even nearer neighbors within Philadelphia, indicating local spread. Multiple genome sequences were available for some subjects and in a subset of cases could be shown to differ between time points and body sites within an individual, indicating heterogeneous viral populations within individuals and raising questions on the mechanisms responsible. There was no evidence that different lineages were associated with different outcomes in patients, emphasizing the importance of individual-specific vulnerability.

SARS-CoV-2 variants associated with vaccine breakthrough in the Delaware Valley through summer 2021.

The severe acute respiratory coronavirus-2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. Evidence suggests that the virus is evolving to allow efficient spread through the human population, including vaccinated individuals. Here we report a study of viral variants from surveillance of the Delaware Valley, including the city of Philadelphia, and variants infecting vaccinated subjects. We sequenced and analyzed complete viral genomes from 2621 surveillance samples from March 2020 to September 2021 and compared them to genome sequences from 159 vaccine breakthroughs. In the early spring of 2020, all detected variants were of the B.1 and closely related lineages. A mixture of lineages followed, notably including B.1.243 followed by B.1.1.7 (alpha), with other lineages present at lower levels. Later isolations were dominated by B.1.617.2 (delta) and other delta lineages; delta was the exclusive variant present by the last time sampled. To investigate whether any variants appeared preferentially in vaccine breakthroughs, we devised a model based on Bayesian autoregressive moving average logistic multinomial regression to allow rigorous comparison. This revealed that B.1.617.2 (delta) showed three-fold enrichment in vaccine breakthrough cases (odds ratio of 3; 95% credible interval 0.89-11). Viral point substitutions could also be associated with vaccine breakthroughs, notably the N501Y substitution found in the alpha, beta and gamma variants (odds ratio 2.04; 95% credible interval of 1.25-3.18). This study thus provides a detailed picture of viral evolution in the Delaware Valley and a geographically matched analysis of vaccine breakthroughs; it also introduces a rigorous statistical approach to interrogating enrichment of viral variants.

SARS-CoV-2 Variants Associated with Vaccine Breakthrough in the Delaware Valley through Summer 2021.

The severe acute respiratory coronavirus-2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. Evidence suggests that the virus is evolving to allow efficient spread through the human population, including vaccinated individuals. Here, we report a study of viral variants from surveillance of the Delaware Valley, including the city of Philadelphia, and variants infecting vaccinated subjects. We sequenced and analyzed complete viral genomes from 2621 surveillance samples from March 2020 to September 2021 and compared them to genome sequences from 159 vaccine breakthroughs. In the early spring of 2020, all detected variants were of the B.1 and closely related lineages. A mixture of lineages followed, notably including B.1.243 followed by B.1.1.7 (alpha), with other lineages present at lower levels. Later isolations were dominated by B.1.617.2 (delta) and other delta lineages; delta was the exclusive variant present by the last time sampled. To investigate whether any variants appeared preferentially in vaccine breakthroughs, we devised a model based on Bayesian autoregressive moving average logistic multinomial regression to allow rigorous comparison. This revealed that B.1.617.2 (delta) showed 3-fold enrichment in vaccine breakthrough cases (odds ratio of 3; 95% credible interval 0.89-11). Viral point substitutions could also be associated with vaccine breakthroughs, notably the N501Y substitution found in the alpha, beta and gamma variants (odds ratio 2.04; 95% credible interval of1.25-3.18). This study thus overviews viral evolution and vaccine breakthroughs in the Delaware Valley and introduces a rigorous statistical approach to interrogating enrichment of breakthrough variants against a changing background. IMPORTANCE SARS-CoV-2 vaccination is highly effective at reducing viral infection, hospitalization and death. However, vaccine breakthrough infections have been widely observed, raising the question of whether particular viral variants or viral mutations are associated with breakthrough. Here, we report analysis of 2621 surveillance isolates from people diagnosed with COVID-19 in the Delaware Valley in southeastern Pennsylvania, allowing rigorous comparison to 159 vaccine breakthrough case specimens. Our best estimate is a 3-fold enrichment for some lineages of delta among breakthroughs, and enrichment of a notable spike substitution, N501Y. We introduce statistical methods that should be widely useful for evaluating vaccine breakthroughs and other viral phenotypes.

Variable levels of drift in tunicate cardiopharyngeal gene regulatory elements.

BACKGROUND: Mutations in gene regulatory networks often lead to genetic divergence without impacting gene expression or developmental patterning. The rules governing this process of developmental systems drift, including the variable impact of selective constraints on different nodes in a gene regulatory network, remain poorly delineated. RESULTS: Here we examine developmental systems drift within the cardiopharyngeal gene regulatory networks of two tunicate species, Corella inflata and Ciona robusta. Cross-species analysis of regulatory elements suggests that trans-regulatory architecture is largely conserved between these highly divergent species. In contrast, cis-regulatory elements within this network exhibit distinct levels of conservation. In particular, while most of the regulatory elements we analyzed showed extensive rearrangements of functional binding sites, the enhancer for the cardiopharyngeal transcription factor FoxF is remarkably well-conserved. Even minor alterations in spacing between binding sites lead to loss of FoxF enhancer function, suggesting that bound trans-factors form position-dependent complexes. CONCLUSIONS: Our findings reveal heterogeneous levels of divergence across cardiopharyngeal cis-regulatory elements. These distinct levels of divergence presumably reflect constraints that are not clearly associated with gene function or position within the regulatory network. Thus, levels of cis-regulatory divergence or drift appear to be governed by distinct structural constraints that will be difficult to predict based on network architecture.