Multiplex CRISPR/Cas9 genome editing in hematopoietic stem cells for fetal hemoglobin reinduction generates chromosomal translocations.

Sickle cell disease and ß-thalassemia are common monogenic disorders that cause significant morbidity and mortality globally. The only curative treatment currently is allogeneic hematopoietic stem cell transplantation, which is unavailable to many patients due to a lack of matched donors and carries risks including graft-versus-host disease. Genome editing therapies targeting either the BCL11A erythroid enhancer or the HBG promoter are already demonstrating success in reinducing fetal hemoglobin. However, where a single locus is targeted, reliably achieving levels high enough to deliver an effective cure remains a challenge. We investigated the application of a CRISPR/Cas9 multiplex genome editing approach, in which both the BCL11A erythroid enhancer and HBG promoter are disrupted within human hematopoietic stem cells. We demonstrate superior fetal hemoglobin reinduction with this dual-editing approach without compromising engraftment or lineage differentiation potential of edited cells post-xenotransplantation. However, multiplex editing consistently resulted in the generation of chromosomal rearrangement events that persisted in vivo following transplantation into immunodeficient mice. The risk of oncogenic events resulting from such translocations therefore currently prohibits its clinical translation, but it is anticipated that, in the future, alternative editing platforms will help alleviate this risk.

AMD3100 redosing fails to repeatedly mobilize hematopoietic stem cells in the nonhuman primate and humanized mouse.

AMD3100 (plerixafor) is a vital component of many clinical and preclinical transplant protocols, facilitating harvest of hematopoietic stem and progenitor cells through mobilization into the peripheral blood circulation. Repeat mobilization with AMD3100 is also necessary for many patients with suboptimal first stem cell collection or those requiring repeat transplantation. In this study we investigated the mobilization efficacy of repeated AMD3100 dosages in the nonhuman primate and humanized mouse models. In nonhuman primates, we observed effective mobilization after the first AMD3100 administration but a significantly poorer response in CD34+ and hematopoietic stem cell-enriched CD90+ cells with subsequent doses of the drug. A similar loss of efficacy with repeated administration was noted in immunodeficient mice engrafted with human CD34+ cells, in whom the total human white cell population, and particularly human hematopoietic stem and progenitor cells, mobilized significantly less effectively following a second AMD3100 administration when compared with the first dose. Together, our results are expected to inform future mobilization protocols for the purposes of peripheral blood hematopoietic stem cell extraction or for applications in which hematopoietic stem cells must be made accessible for in vivo-delivered gene targeting agents.

Therapeutically relevant engraftment of a CRISPR-Cas9-edited HSC-enriched population with HbF reactivation in nonhuman primates.

Reactivation of fetal hemoglobin (HbF) is being pursued as a treatment strategy for hemoglobinopathies. Here, we evaluated the therapeutic potential of hematopoietic stem and progenitor cells (HSPCs) edited with the CRISPR-Cas9 nuclease platform to recapitulate naturally occurring mutations identified in individuals who express increased amounts of HbF, a condition known as hereditary persistence of HbF. CRISPR-Cas9 treatment and transplantation of HSPCs purified on the basis of surface expression of the CD34 receptor in a nonhuman primate (NHP) autologous transplantation model resulted in up to 30% engraftment of gene-edited cells for >1 year. Edited cells effectively and stably reactivated HbF, as evidenced by up to 18% HbF-expressing erythrocytes in peripheral blood. Similar results were obtained by editing highly enriched stem cells, defined by the markers CD34+CD90+CD45RA-, allowing for a 10-fold reduction in the number of transplanted target cells, thus considerably reducing the need for editing reagents. The frequency of engrafted, gene-edited cells persisting in vivo using this approach may be sufficient to ameliorate the phenotype for a number of genetic diseases.

High prevalence of cardiovascular and respiratory abnormalities in advanced, intensively treated (transplanted) myeloma: The case for 'late effects' screening and preventive strategies.

OBJECTIVES: Modern management of myeloma has significantly improved survival, with increasing numbers of patients living beyond a decade. However, little is known about the long-term cardiovascular and respiratory status of intensively treated and multiply relapsed survivors. METHODS: We performed detailed cardiovascular and respiratory evaluations in patients with intensively treated, advanced but stable myeloma. All patients had received at least two lines of treatment, including at least one haematopoietic stem cell transplantation procedure, but had stable, controlled disease and were off active treatment at the time of evaluation. RESULTS: Thirty-two patients with a median duration of 6 years (range 2-12) from original diagnosis of myeloma and three lines (range 2-6) of treatment were evaluated. Despite normal physical examination in the majority, there was a high prevalence of sub-clinical cardiac and respiratory dysfunction, reflected by abnormalities of electrocardiography (45%), echocardiography (50%), serum N-terminal pro-B-type natriuretic peptide level (NT-pro-BNP, 50%), and pulmonary function testing (45%). NT-pro-BNP level correlated negatively with quality of life (P = 0.012) and positively with serum ferritin (P = 0.027). Dyspnoea score correlated with BMI (P = 0.001). Risk factors for cardiovascular disease (obesity, hypertension, hyperlipidaemia, and hyperinsulinaemia) were common. DISCUSSION: Even in the absence of overt clinical features, the majority of intensively treated long-term survivors of myeloma have established cardiovascular and/or respiratory dysfunction, above levels expected in the general population of a similar age. CONCLUSION: This study supports routine screening and lifestyle modification combined with primary and secondary preventive strategies to reduce cardiovascular and respiratory disease and to preserve quality of life in transplanted myeloma patients.

The impact of insulin resistance on the outcome of laparoscopic ovarian diathermy in women with polycystic ovarian syndrome.

OBJECTIVE: Insulin resistance (IR), which is common in women with polycystic ovarian syndrome (PCOS), may adversely affect the outcome of ovulation induction due to its detrimental effect on ovarian function. The aim of this study was to evaluate the impact of IR on the outcome of laparoscopic ovarian diathermy (LOD). DESIGN: Prospective observational study. SETTING: Tertiary fertility centre. POPULATION: Forty-four anovulatory women with PCOS undergoing LOD. METHODS: Fasting serum concentrations of insulin and glucose were measured before and after LOD. Insulin sensitivity was determined using the homeostasis model assessment (HOMA) index. Values of HOMA index ≥2.1 were considered abnormal, indicating IR. MAIN OUTCOME MEASURES: Ovulation and pregnancy rates were compared between women with and without IR using χ(2) test and odds ratio (OR) with 95% confidence interval (CI). RESULTS: Eighteen women (41%) had elevated HOMA index (≥2.1). There was no change of HOMA index after LOD (median 1.89, range 0.53-8.48 vs. 1.92, range 0.53-8.46). The ovulation rate in women with IR (12 of 18, 67%) was significantly (p=0.013, OR 0.08; 95% CI 0.01-0.74) lower than that (25 of 26, 96%) of women without IR. Likewise, the pregnancy rate (six of 18, 33%) was significantly (p=0.037, OR 0.26; 95% CI 0.07-0.94) lower in women with IR than that of women without IR (17 of 26, 65%). Using a receiver operating characteristics curve, the HOMA index was useful in predicting no ovulation after LOD (area under the curve 0.826). CONCLUSIONS: Insulin resistance appears to have an adverse effect on the outcome of LOD and seems to be a useful prognostic factor.

Idiopathic thrombocytopenic purpura associated with an astrocytoma.

We present the case of an 8-year-old girl with chronic idiopathic thrombocytopenic purpura (ITP) and a short history suggestive of raised intracranial pressure. Urgent computed tomography scan of the head showed a large bleed into a left cerebellar lesion. She underwent treatment with intravenous immunoglobulin (IVIg) and steroids to increase her platelet count, followed by excision of the lesion, which was found to be a benign pilocytic astrocytoma. The patient made a complete recovery and shortly afterwards underwent splenectomy, following which there was complete resolution of her thrombocytopenia.