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Alpha thalassemia major (ATM) is a hemoglobinopathy that usually results in perinatal demise if in utero transfusions (IUTs) are not performed. We established an international registry (NCT04872179) to evaluate the impact of IUTs on survival to discharge (primary outcome) as well as perinatal and neurodevelopmental secondary outcomes. Forty-nine patients were diagnosed prenatally, 11 were diagnosed postnatally, and all 11 spontaneous survivor genotypes had preserved embryonic zeta-globin levels. We compared 3 groups of patients; group 1, prenatally diagnosed and alive at hospital discharge (n = 14), group 2, prenatally diagnosed and deceased perinatally (n = 5), and group 3, postnatally diagnosed and alive at hospital discharge (n = 11). Group 1 had better outcomes than groups 2 and 3 in terms of the resolution of hydrops, delivery closer to term, shorter hospitalizations, and more frequent average or greater neurodevelopmental outcomes. Earlier IUT initiation was correlated with higher neurodevelopmental (Vineland-3) scores (r = −0.72, P = .02). Preterm delivery after IUT was seen in 3/16 (19%) patients who continued their pregnancy. When we combined our data with those from 2 published series, patients who received ≥2 IUTs had better outcomes than those with 0 to 1 IUT, including resolution of hydrops, delivery at ≥34 weeks gestation, and 5-minute appearance, pulse, grimace, activity, and respiration scores ≥7. Neurodevelopmental assessments were normal in 17/18 of the ≥2 IUT vs 5/13 of the 0 to 1 IUT group (OR 2.74; P = .01). Thus, fetal transfusions enable the survival of patients with ATM and normal neurodevelopment, even in those patients presenting with hydrops. Nondirective prenatal counseling for expectant parents should include the option of IUTs.

Introduction: Alpha globin mutations are very common worldwide, and the severity of resulting anemia depends on the number and type of mutated alleles. While the 4 gene mutation (alpha thalassemia major, ATM) was previously deemed fatal except in rare cases, emerging evidence indicates that survival to birth and good postnatal outcomes are possible with in utero transfusions. We hypothesized that the embryonic zeta globin gene, which is expressed early in gestation prior to alpha globin, may compensate for the lack of alpha globin and that induction of zeta globin after it has naturally been silenced may become a new therapy for patients with ATM. Methods: We evaluated mutations in the UCSF international registry of patients with ATM to understand factors related to patient survival with and without in utero transfusions. We then engineered Human Umbilical Cord Derived Erythroid Progenitor Cells (HUDEP-2 cells) carrying the common SEA alpha globin deletion, in which zeta globin expression is preserved (H-SEA), as well as those on which the zeta globin genes were deleted (HBZ-/-) using CRISPR-Cas9. We evaluated the expression of alpha and zeta globins using qPCR, Western blot, and flow cytometry. We generated lentiviral vectors expressing zeta globin under the control of beta-globin promoters to examine changes in both zeta and alpha globin in a dynamic way. Results: None of the registry patients who survived to birth spontaneously (n=11) had a mutation that involves a concomitant deletion in zeta globin (such as the -FIL, -THAI, or -MEDII mutation), while alpha globin mutations extending into the zeta globin gene were found in 14 of 37 (38%) patients who were diagnosed prenatally, suggesting that the presence of zeta globin may play a role in the ability to survive to birth without fetal therapy. Interestingly, we found that H-SEA clones express higher levels of zeta globin than WT cells, as illustrated by quantitative real-time PCR (Fig 1A), Western blot (Fig 1B) and flow cytometry (Fig 1C). These cells also developed beta globin dimers due to excess unpaired beta-globin chains, as demonstrated by Western blotting with and without reducing agents, indicating that they are an appropriate cell model for ATM. We next generated HUDEP-2 clones lacking zeta globin (HBZ KO) and transduced these clones with lentiviral vectors expressing high levels of zeta globin (lenti-zeta) (Fig 1D). Western blotting revealed that increasing the levels of zeta globin in these cells resulted in decreased expression of alpha globin, suggesting reciprocal control between these genes (Fig 1E). Most importantly, we saw a reduction in toxic beta-globin dimers in H-SEA cells expressing high levels of zeta-globin after transduction with lenti-zeta, suggesting that zeta globin could functionally replace the missing alpha-globin (Fig 1 F,G). To understand transcriptomic differences in H-SEA cells that may result in increased zeta globin expression, we performed bulk RNA sequencing of wild type and H-SEA clones. We confirmed that zeta expression is significantly upregulated in H-SEA compared to wild type (log2 fold change of 4.25, p=2.24E-38). Pathway analysis of differentially expressed genes is ongoing. Conclusions: Our international patient registry suggests that expression of zeta globin may play a role in the spontaneous survival to birth in a subset of patients. Zeta globin expression is increased in the setting of H-SEA cells in vitro, and restoration of zeta expression by lentivirus results in a reduction of toxic beta globin dimers in these ATM cells. Furthermore, expressing zeta globin at high levels in H-WT cells decreased alpha globin expression, suggesting a reciprocal regulation of these two genes. This concept is similar to the relationship between fetal gamma and adult beta globins which has been exploited for therapeutic editing approaches in patients with beta-thalassemia. At this point, the natural repressor of zeta globin is not yet known, but our data suggests that a strategy of upregulating zeta globin could potentially be developed to mimic the ongoing trials of using the BCL11A repressor to induce gamma globin in patients with beta thalassemia and sickle cell disease. Disclosures Wienert: Integral Medicines: Current Employment. Kohn:Allogene Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Patents & Royalties, Research Funding. MacKenzie:Acrigen: Membership on an entity's Board of Directors or advisory committees; Ultragenyx: Research Funding.