Fanconi Anemia Patients Research Articles

Compromised Host Stem Cell Competitiveness Affords Fanconi Stem Cell Engraftment in C-Kit Mutant Humanized Mice

Humanized mouse model is a powerful tool for the study of human hematopoiesis and hematologic diseases in vivo. However, existing models continue to be limited by absent engraftment of stem cells with inherent growth defects, especially in bone marrow failure (BMF) disorders such as low-grade myelodysplastic syndromes (MDS) and Fanconi Anemia (FA). Previously, we presented a highly efficient MDS/AML PDX model in cytokine-humanized MISTRG mice, that express human M-CSF, IL-3, GM-CSF and THPO in the background of the SIRPα - humanized rag -/- IL2Rg -/- mice. MISTRG mice support efficient, faithful and long-term MDS hematopoietic stem and progenitor cell (HSPC) engraftment with multi-lineage representation. Similar to MDS, FA stem cells are defective and sensitive to toxic stress conferred by inflammation or irradiation. Irradiation, required in conventional mice as pre-conditioning, increases the risk of secondary malignancies, induces activation of inflammatory pathways and may damage the bone marrow microenvironment.In this study, we present MISTRG6kit W41 mice that in addition to the above cytokines express human IL-6, which improves lymphoid development, and that were CRISPR modified to introduce the white spotting 41 mutation in c-Kit (c-kit W41), which results in partial loss of c-kit function in the murine host stem and progenitor cells. To characterize this mouse model, we compare the engraftment capacity between MISTRG6 and MISTRG6kit W41 mice. We find that the HSPCs from both healthy donor and low grade MDS patient can be more efficiently propagated in MISTRG6Kit W41 mice and that they can be serially transplanted. Human cells are detectable at higher levels in PB of MISTRG6Kit W41 mice as early as 8 weeks post transplantation. As previously described, c-kit mutation enhances human erythroid engraftment while multi-lineage engraftment is maintained in engrafted MISTRG6Kit W41 mice. Therefore, MISTRG6Kit W41 mice offer a better host environment that supports human hematopoietic expansion without irradiation.FA is an inherited genetic disease carrying FA gene mutations associated with DNA damage and cancer predisposition and marked by BMF, MDS and AML. FA HSPCs cannot be expanded or grown for sufficient periods in vitro, and existing mouse models have failed to demonstrate engraftment of primary cells from FA patient. We transplanted a MDS sample from a FANCC mutant patient into MISTRG6Kit W41 mice giving rise to a myeloid predominant graft that could be successfully serially transplanted. Moreover, non-transformed FA HSPCs from 2 FANCA and 2 FANCG mutant patients were successfully engrafted in MISTRG6Kit W41 mice.In summary, MISTRG mice with human IL-6 expression and c-Kit W41 mutation provide a more efficient and convenient way for pre-clinical studies of BMFs such as FA and low-grade MDS with maintenance of an intact humanized stem cell niche, and this is the first time we saw primary cells from FA patient propagate in mouse model. We believe that our model would be highly beneficial to the study of additional diseases associated with DNA damage or cancer predisposition, such as dyskeratosis congenita or Schwachman-Diamond syndrome. DisclosuresNo relevant conflicts of interest to declare.

Mitotic Errors Promote Genomic Instability and Leukemia in a Novel Mouse Model of Fanconi Anemia.

Fanconi anemia (FA) is a disease of genomic instability and cancer. In addition to DNA damage repair, FA pathway proteins are now known to be critical for maintaining faithful chromosome segregation during mitosis. While impaired DNA damage repair has been studied extensively in FA-associated carcinogenesis in vivo, the oncogenic contribution of mitotic abnormalities secondary to FA pathway deficiency remains incompletely understood. To examine the role of mitotic dysregulation in FA pathway deficient malignancies, we genetically exacerbated the baseline mitotic defect in Fancc-/- mice by introducing heterozygosity of the key spindle assembly checkpoint regulator Mad2. Fancc-/-;Mad2+/- mice were viable, but died from acute myeloid leukemia (AML), thus recapitulating the high risk of myeloid malignancies in FA patients better than Fancc-/-mice. We utilized hematopoietic stem cell transplantation to propagate Fancc-/-; Mad2+/- AML in irradiated healthy mice to model FANCC-deficient AMLs arising in the non-FA population. Compared to cells from Fancc-/- mice, those from Fancc-/-;Mad2+/- mice demonstrated an increase in mitotic errors but equivalent DNA cross-linker hypersensitivity, indicating that the cancer phenotype of Fancc-/-;Mad2+/- mice results from error-prone cell division and not exacerbation of the DNA damage repair defect. We found that FANCC enhances targeting of endogenous MAD2 to prometaphase kinetochores, suggesting a mechanism for how FANCC-dependent regulation of the spindle assembly checkpoint prevents chromosome mis-segregation. Whole-exome sequencing revealed similarities between human FA-associated myelodysplastic syndrome (MDS)/AML and the AML that developed in Fancc-/-; Mad2+/- mice. Together, these data illuminate the role of mitotic dysregulation in FA-pathway deficient malignancies in vivo, show how FANCC adjusts the spindle assembly checkpoint rheostat by regulating MAD2 kinetochore targeting in cell cycle-dependent manner, and establish two new mouse models for preclinical studies of AML.

Gene Therapy for Fanconi Anemia [Group A]: Interim Results of RP-L102 Clinical Trials

Gene Therapy for Fanconi Anemia [Group A]: Interim Results of RP-L102 Clinical Trials

Novel Ubiquitinated Proteins Downstream of the Fanconi Anemia Core Complex

The Fanconi anemia (FA) pathway is a major player in the control of DNA replication integrity in response to replication stress. Germline defect in the pathway results in the FA syndrome characterized by developmental abnormalities, bone marrow (BM) failure, and genome instability which greatly elevates the incidence of cancers. A pivotal step in the activation of the FA DNA repair pathway is the monoubiquitination of the FANCD2 and FANCI proteins (ID2) by the FA core complex, a unique ubiquitin ligase complex which includes eight proteins (FANCA-FANCG, FANCL, and FAAP100) and UBE2T/FANCT. This monoubiquitination event enables the recruitment of the ID2 complex to chromatin and nuclear foci at sites of DNA damage. Cells with mutations in any of the FA core complex proteins lack the ability to monoubiquitinated ID2, making ID2 ubiquitination a convergence point in the pathway, with an estimation of>90% FA patients defective in this step. Additionally, somatic mutations In FA genes render tumor cells sensitive to DNA crosslinking agents, so identification of FA pathway defects provides an opportunity for therapeutic targeting. In search for additional potential target/substrate of this unique FA core ubiquitin ligase complex, we performed a high throughput genome-wide ubiquitin-specific proteomics (UbiScan) screen and found, in addition to the ID2 complex, many ubiquitinated proteins are dysregulated (mostly downregulated) in FA deficient cells compared with that of FA proficient cells.We used a Ubiquitin Remnant Motif (K- ∑-GG) Antibody Bead Conjugate (Cell Signaling Technology), a proprietary ubiquitin branch (“K- ∑-GG”) antibody with specificity for a di-glycine tag that is the remnant of ubiquitin left on protein substrates after trypsin digestion, to enrich ubiquitinated peptides from trypsin digested cell samples (shNT vs shFANCA). This enrichment is followed by LC-MS/MS analysis for quantitative profiles of hundreds to over a thousand nonredundant ubiquitinated sequences. We were successful in demonstrating that under steady-state conditions (without proteasome inhibitor treatment), the ubiquitinated forms of both FANCD2 and FANCI proteins are much higher in control (shNT) HeLa cells compared with that of the cells depleted of FANCA (shFANCA).We then collaborated with the Cell signaling technology to perform a high throughput UbiScan® analysis of total ubiquitinated proteins both in total nuclei and chromatin fractions under replicative stress conditions. UbiScan® enables researchers to isolate, identify and quantitate large numbers of ubiquitin-modified cellular peptides with a high degree of specificity and sensitivity, providing a global overview of the ubiquitination sites in cellular proteins in cell and tissue samples without preconceived biases about where these modified sites occur. A total of 16,249 redundant modified peptide assignments to 7,856 modified sites for the Ubiquitin K-GG Remnant Motif Antibody were obtained. As expected, the amount of monoubiquitinated FANCD2 (at K651) and FANCI (at K523) were highly reduced in both the nuclear and chromatin fractions of Hela cells depleted of FANCA (shA). Consistent with the earlier findings, the amount of ubiquitinated ID2 proteins were extremely low in the chromatin fraction of the Hela cells depleted of FANCA.Since there are numerous ubiquitinated proteins found to be dysregulated in our UbiScan analyses, we used the following criteria to select the target proteins based on; a) -fold changes, and b) proteins that are known to participate in the DNA repair signaling pathways. We validated our UbiScan results by using an assay system to detect endogenous protein ubiquitination. We also found a significant reduction in the ubiquitination of several DNA repair-related proteins (found in our UbiScan analysis) in FANCA deficient cells. To assess FA pathway functions, we generated HAP1 and appropriate cells knock out of these select ubiquitinated target proteins by using CRISPR-Cas9 system. Then, the KO cells were examined for FA pathway functions. These results will be discussed.In conclusion, our findings reveal that the FA core ubiquitin ligase complex regulates (directly or indirectly) the ubiquitinated levels of many novel proteins outside of the ID2 complex, and these novel target proteins may provide important additional mechanistic insights into the FA DNA repair pathway. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of PARP1 Reduces Cell Viability and Increases Markers of DNA Damage in Fanconi Anemia-Mutated Acute Myeloid Leukemia

Introduction:Fanconi Anemia (FA) is a hereditary disorder characterized by deficiencies in DNA damage repair and genome instability with a high propensity for bone marrow failure (BMF) and malignancies such as acute myeloid leukemia (AML). Clinically, FA patients experience greater toxicity than non-FA patients when treated with cytotoxic chemotherapy used for AML treatment, so there is a need for alternative treatments to be developed for FA-mutated AML.Poly (ADP-ribose) polymerase 1 (PARP1) is an important enzyme involved in the recognition and repair of DNA breaks. There has been recent clinical success in treating cancers with defective DNA damage repair with PARP inhibitors, an example of synthetic lethality. Therefore we hypothesize that PARP inhibition (PARPi) is an effective strategy for treating FA-mutated AML. Recent studies have shown that PARP1 is overexpressed in many cancers, including AML, and that higher PARP1 expression is associated with worse patient outcomes. Here, we investigate the anti-tumor effects of a PARP inhibitor, olaparib, on FA-mutated and wild-type (WT) AML cells and investigate the activity of downstream DNA repair pathways in response to PARPi.Methods/Results:To determine the effects of PARPi on AML and FA-mutated AML cells in vitro, we treated four cell lines, one FA-wild type AML line and three patient-derived FA-mutated AML lines, with olaparib for 1, 4, 8, 24, and 48 hours. Preliminary data suggest that olaparib treatment decreases protein expression of both PARP1 and PAR (from activation of PARP) compared to vehicle controls.To evaluate the effect of PARPi on DNA damage in AML we measured γH2AX expression by western blotting and immunofluorescence, and found that, although γH2AX expression was not significantly increased in FA-wild type AML cells, there was a significant increase in γH2AX expression in SB1685 FA-mutated AML cells treated with olaparib compared to controls after 4 hours of treatment (p-value < 0.05).To further evaluate the ability of olaparib to inhibit DNA damage repair, we treated our cells with olaparib and performed single-cell alkaline electrophoresis COMET assay. We found that, while the WT cell line was able to repair its DNA over time (indicated by lower levels of DNA damage after 48 hours of olaparib exposure compared to earlier time points), our FA-mutated AML cell lines had more DNA damage after 48 hours of treatment compared to controls. These data suggest that, while cells proficient in DNA repair are capable of repairing DNA damage even when exposed to PARPi, cells that have mutations in their ability to repair DNA damage are not only less able to repair DNA damage over time but also show increased DNA damage over time when exposed to PARPi.To better understand the effects of this increase in DNA damage, we treated our cells with olaparib and assayed for cell viability over 96 hours. We found that, while WT AML cells did not have significantly decreased cell viability after 96 hours, FA-mutated cell lines trended towards significant decrease in cell viability at 96 hours. These cell lines were also stained with Annexin V to investigate apoptotic activity. Our results indicate that olaparib is able to induce apoptosis in our FA-mutated cells after 24 hours of treatment and that, as treatment continues, the percent of Annexin V-positive cells increases compared to controls.To investigate downstream DNA damage response to PARPi, we treated our cells with olaparib and analyzed the expression of DNA Ligase III, Mre11, XRCC1, and Rad51-enzymes involved in various DNA repair pathways. We found that expression levels of XRCC1 increased over 48 hours in our WT AML cells, suggesting a response to the DNA damaging effects of PARPi. In our FA-mutated SB1685 cells, we found a decrease in XRCC1, DNA Ligase III, and Rad51. The expression levels of these enzymes in the other FA-mutated cell lines were more variable, suggesting that the impact of PARPi on downstream DNA repair pathways may be different across different cell lines.Conclusions:Our data suggest that PARP inhibition may be a potential therapy for the treatment of acute myeloid leukemia. In particular, leukemia with mutations in DNA repair mechanisms may be more responsive to PARP inhibition due to resulting DNA damage and synthetic lethality. Thus, PARP inhibitors have the potential to be an effective therapeutic strategy for the treatment of FA-mutated AML. DisclosuresNo relevant conflicts of interest to declare.

INVESTIGATION OF SALIVARY miR-9, miR-34a ve miR-196a LEVELS IN FANCONI ANEMIA AND ORAL SQUAMOUS CELL CARCINOMA PATIENTS

Fanconi anemia (FA) is a rare bone marrow deficiency syndrome due to the DNA repair gene mutations, and Oral Squamous Cell Carcinoma (OSCC) is seen more frequently in FA patients than in the general population. The dysregulation of PI3K and Wnt signaling has been implicated in OSCC pathogenesis and abnormal expressions of miRNAs (a class of non-coding small regulatory RNAs) associated with these signaling pathways has been reported in OSHK patients. Salivary miRNAs are valuable biomarker candidates for OSCC development and prognosis. In this study, salivary levels of miR-9, miR-34a and miR-196a miRNAs related to PI3K and Wnt signaling pathways were examined in OSCC and FA patients and compared with the healthy control group. Saliva samples were collected from 89 subjects including 25 OSCC patients, 24 FA patients and 40 healthy controls. Total RNA was isolated using Quick-RNA Miniprep Kit (Zymo Research) due to the kit instructions. cDNA was generated with miRCURY LNA miRNA PCR Assay (Qiagen, Hilden, Germany) and Quantitative real-time PCR was performed with miRCURY LNA SYBR Green PCR Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. For the normalization of the expression levels of each miRNA, the mean expression U6 SnRNA was used as reference. The ΔΔCt value and the normalized miR-9, miR-34a and miR-196a salivary levels were calculated with Livak Method. Our results showed that miR-9 and miR-34a levels in OSCC patients were significantly lower compared to healthy control groups (p= 0,01 and p= 0,012), and there was no significant difference in miR-196a levels (p&gt; 0,05). In FA patients, miR-9 and miR-34 levels were lower than in control groups, likewise the OSCC patients (p =0,017 and p =0,014). There was no significant difference between miR-9, miR-34a, and miR-196a levels of FA patients and OSCC patients (p &gt;0.05). According to our results, low levels of miR-9 and miR-34a in saliva are biomarker candidates that may be important for OSCC development. In FA patients, close follow-up of the levels of miR-9 and miR-34 would be appropriate considering OSCC development. Further studies are needed to confirm the potential of miR-9 and miR-34a as biomarkers for OSCC.

A comprehensive molecular study identified 12 complementation groups with 56 novel FANC gene variants in Indian Fanconi anemia subjects.

Fanconi anemia (FA) is a rare autosomal or X-linked genetic disorder characterized by chromosomal breakages, congenital abnormalities, bone marrow failure (BMF), and cancer. There has been a discovery of 22 FANC genes known to be involved in the FA pathway. This wide number of pathway components makes molecular diagnosis challenging for FA. We present here the most comprehensive molecular diagnosis of FA subjects from India. We observed a high frequency (4.42 ± 1.5 breaks/metaphase) of chromosomal breakages in 181 FA subjects. The major clinical abnormalities observed were skin pigmentation (70.2%), short stature (46.4%), and skeletal abnormalities (43.1%), along with a few minor clinical abnormalities. The combination of Sanger sequencing and Next Generation Sequencing could molecularly characterize 164 (90.6%) FA patients and identified 12 different complementation groups [FANCA (56.10%), FANCG (16.46%), FANCL (12.80%), FANCD2 (4.88%), FANCJ (2.44%), FANCE (1.22%), FANCF (1.22%), FANCI (1.22%), FANCN (1.22%), FANCC (1.22%), FANCD1 (0.61%) and FANCB (0.61%)]. A total of 56 novel variants were identified in our cohort, including a hotspot variant: a deletion of exon 27 in the FANCA gene and a nonsense variant at c.787 C>T in the FANCG gene. Our comprehensive molecular findings can aid in the stratification of molecular investigation in the diagnosis and management of FA patients.

Kidney complications in 107 Fanconi anemia patients submitted to hematopoietic cell transplantation

Fanconi anemia (FA) is a rare disease characterized by progressive bone marrow failure, cancer predisposition, and multiple systemic malformations, including congenital abnormalities of the kidney and urinary tract (CAKUT). Hematopoietic cell transplantation (HCT), the only potentially curative treatment for the hematological complications of FA, may precipitate acute kidney injury (AKI) and hypertension. We retrospectively investigated 107 FA patients who underwent HCT between 2009 and 2017. We investigated the incidence and risk factors of AKI within 100 days after HCT in a cohort of FA patients, and kidney function and hypertension over 2-year follow-up.The incidence of AKI (mainly stage I) was 18.7%. Patients aged ≥ 11 years at transplantation showed a higher risk of AKI (OR 3.53). The eGFR was 60-90 mL/min/1.73 m2 in 53 (49.5%), 55 (51.4%), 50 (50.5%), 50 (51%), and 46 (59.7%) patients before HCT, at 100 days, 6 months, 1 year, and 2 years. Within the first 100 days after HCT, hypertension was observed in 72% of the patients and was associated with cyclosporine therapy. Most (62.3%) patients had stage 2 hypertension. CAKUT was observed in 33.7% of the patients and was associated with both hypertension (86%) and diminished kidney function but not with AKI.Conlusion: Although AKI, a commonly known HCT complication, was mild in this study, the prevalence of chronic kidney disease (CKD), as well as the high incidence of hypertension, specially associated with CAKUT point out the importance of kidney care in short and long-term follow up of FA patients. What is Known: • Fanconi anemia (FA) is the most frequent inherited bone marrow failure in children, and 30% of cases have congenital anomalies of kidney (CAKUT). • Acute kidney injury and hypertension after hematopoietic cell transplantation (HCT) may impact the outcomes.. What is New: • Despite the presence of CAKUT and stage 2 CKD in 33.7% and 50% of the patients, respectively, AKI was mild and transitory after HCT in FA patients. • CAKUT in FA patients was associated with lower kidney function and hypertension after HCT.

Post-hematopoietic stem cell transplant squamous cell carcinoma in patients with Fanconi anemia: a dreadful enemy.

Hematopoietic stem cell transplantation (HSCT) is a curative option for patients with Fanconi anemia (FA) and hematological manifestations but it does not prevent solid tumors, especially squamous cell carcinomas (SCC). Retrospective study in 22 FA patients who had received HSCT and had been followed up beyond 2years after HSCT. The median follow-up was 15years. Six patients developed head-and-neck SCC after transplantation. The cumulative incidence of SCC at 15 and 30years from the HSCT was 14.2% and 71.2%, respectively. One patient was diagnosed in stage IV and the rest, who were being followed up in cancer screening programs, in stage I. Treatment of SCC consisted of surgery in all patients; radiotherapy and chemotherapy were used in two patients and were poorly tolerated. FA patients have high risk of head-and-neck SCC. Multi-disciplinary programs for early cancer detection are of special relevance in these patients.

FANCD2 limits acetaldehyde-induced genomic instability during DNA replication in esophageal keratinocytes.

Individuals with Fanconi anemia (FA), a rare genetic bone marrow failure syndrome, have an increased risk of young‐onset head and neck squamous cell carcinomas (SCCs) and esophageal SCC. The FA DNA repair pathway is activated upon DNA damage induced by acetaldehyde, a chief alcohol metabolite and one of the major carcinogens in humans. However, the molecular basis of acetaldehyde‐induced genomic instability in SCCs of the head and neck and of the esophagus in FA remains elusive. Here, we report the effects of acetaldehyde on replication stress response in esophageal epithelial cells (keratinocytes). Acetaldehyde‐exposed esophageal keratinocytes displayed accumulation of DNA damage foci consisting of 53BP1 and BRCA1. At physiologically relevant concentrations, acetaldehyde activated the ATR‐Chk1 pathway, leading to S‐ and G2/M‐phase delay with accumulation of the FA complementation group D2 protein (FANCD2) at the sites of DNA synthesis, suggesting that acetaldehyde impedes replication fork progression. Consistently, depletion of the replication fork protection protein Timeless led to elevated DNA damage upon acetaldehyde exposure. Furthermore, FANCD2 depletion exacerbated replication abnormalities, elevated DNA damage, and led to apoptotic cell death, indicating that FANCD2 prevents acetaldehyde‐induced genomic instability in esophageal keratinocytes. These observations contribute to our understanding of the mechanisms that drive genomic instability in FA patients and alcohol‐related carcinogenesis, thereby providing a translational implication in the development of more effective therapies for SCCs.

FANCI functions as a repair/apoptosis switch in response to DNA crosslinks.

Cells counter DNA damage through repair or apoptosis, yet a direct mechanism for this choice has remained elusive. When facing interstrand crosslinks (ICLs), the ICL-repair protein FANCI heterodimerizes with FANCD2 to initiate ICL excision. We found that FANCI alternatively interacts with a pro-apoptotic factor, PIDD1, to enable PIDDosome (PIDD1-RAIDD-caspase-2) formation and apoptotic death. FANCI switches from FANCD2/repair to PIDD1/apoptosis signaling in the event of ICL-repair failure. Specifically, removing key endonucleases downstream of FANCI/FANCD2, increasing ICL levels, or allowing damaged cells into mitosis (when repair is suppressed) all suffice for switching. Reciprocally, apoptosis-committed FANCI reverts from PIDD1 to FANCD2 after a failed attempt to assemble the PIDDosome. Monoubiquitination and deubiquitination at FANCI K523 impact interactor selection. These data unveil a repair-or-apoptosis switch in eukaryotes. Beyond ensuring the removal of unrepaired genomes, the switch's bidirectionality reveals that damaged cells can offset apoptotic defects via de novo attempts at lesion repair.

Bone Marrow Failure Disease and Head-and-Neck Squamous Cell Carcinoma in King Faisal Specialist Hospital and Research Center in Riyadh

Objective: This study aims to look at bone marrow failure disease patients who presented to our center, as to analyze the presentation and possible ways to improve the survival outcome in these cases. Methods: This was a retrospective chart review study. Results: In our series, most of the head-and-neck squamous cell carcinomas (HNSCCs) were found in Fanconi anemia (FA) patients, and most are located within the oral cavity, most frequently at the tongue margins and the gingival areas. These tumors arise in both male and female bone marrow failure disease patients without a prior history of excessive tobacco and alcohol use. Even if a patient presents at an early stage, there are frequent relapses. The highly aggressive nature of the tumors is reflected by poor survival, amounting to &lt;2 years. FA is a rare but well-studied inherited disorder that is clinically characterized by progressive bone marrow failure, congenital malformations, and increased incidence of malignancies, especially acute myeloid leukemia and squamous cell carcinomas (SSCs) of the head and neck. Greatly improved protocols for stem cell transplantation increasingly save the lives of these young patients. However, in both transplanted and not transplanted patients, the emergence of aggressive SSC represents a major medical challenge. Noninvasive screening options, including frequent self-examination and inspection by a medical team, should lead to early detection and treatment. Standard platinum-based chemotherapy in combination with radiotherapy cannot be used in FA patients due to therapy-associated toxicities and mortalities even with reduced dosing. Therefore, surgery is the most important treatment option for HNSCC in bone marrow failure disease patients and requires an early and efficient detection of malignant lesions. Conclusion: So far, no uniform treatment protocol for the management of HNSCCs in FA patients exists. Therefore, we propose that the information on affected FA patients should be collected worldwide, practical therapeutic guidelines developed and national treatment centers established.

Excellent Outcome of Alternative Donor HSCT in Fanconi Anemia Using Chemotherapy Only Regimen: Single Korean Institution Experience

Title) Excellent outcome of alternative donor HSCT in Fanconi anemia using chemotherapy only conditioning regimen: single Korean institution experience.Introduction) Fanconi anemia (FA) is a rare autosomal (or X linked) recessive disorder characterized by bone marrow failure, secondary malignancies, and skeletal anomalies. Hematopoietic stem cell transplantation (HSCT) remains the standard treatment of hematologic disease. Although HSCT from a matched sibling donor (MSD) is the optimal mode of transplant, many patients lack such a donor necessitating alternative donor HSCT (AD-HSCT). Radiation treatment during the conditioning regimen results in a high incidence of post-HSCT complications in FA patients, while serotherapy with alemtuzumab has been reported to increase infectious toxicities. Here, we report on the excellent outcome of 15 FA patients who received AD-HSCT with a fludarabine-based conditioning regimen with in vivo T cell depletion using rabbit anti-thymocyte globulin (ATG).Methods and Results) We reviewed the medical records of FA patients diagnosed by chromosomal breakage test and who received AD-HSCT between September 2007 and December 2016. Median age at transplant was 10.8 years (4.2~18.6) and 7 were male (46.7%). Eleven of 14 patients who received genetic study for FA were found to have mutations (6 with FANC-A mutations (40%), and 5 with FANC-G mutations (33.3%)). Hematologic reasons for HSCT were severe aplastic anemia in 13 (93.3%) and myelodysplastic syndrome in 2 patients (13.3%). One patient received HSCT from a mismatched related donor, while the remaining patients received unrelated HSCT. Seven transplants were human leukocyte antigen (HLA) fully matched (13.3%). Cell sources were as follows: peripheral blood stem cells 9 (60%), cord blood 4 (26.7%), bone marrow 2 (13.3%). Conditioning regimen consisted of fludarabine 30mg/BSA/day x5days (D-8,-7,-6,-5,-4), cyclophosphamide 5mg/kg/day x4days (D-5,-4,-3,-2), rabbit ATG 2.5mg/kg/day x3days (D-3,-2,-1). Graft-versus-host disease prophylaxis consisted of cyclosporine and mini-dose methotrexate. Medians of infused cell doses were as follows: TNC 16.51 x108/kg (5.17~42.4), CD34+ 15.49x106/kg(3.49~33.7).Twelve patients (80%) showed initial engraftment and full donor chimerism according to short tandem repeat polymerase chain reaction (STR-PCR) analysis. Of the 3 patients who showed primary graft failure, one showed autologous recovery, while the remaining 2 received additional HSCs from the same donor and a haploidentical family donor respectively, leading to engraftment. Acute GVHD grade II or above was diagnosed in 8 patients (53.5%) and resolved with steroid therapy. Chronic GVHD developed in two patients, one mild and one of moderate severity. The patient with moderate oral chronic GVHD was diagnosed with squamous cell carcinoma of the tongue 5years after HSCT. She received surgical resection and remains disease-free for 40 months. After transplantation, 5 patients (33.3%) experienced infectious complications including viral reactivation, bacteremia and fungemia but all recovered without complications. At a median follow-up of 59.6 months (6.3~107.9) all 15 FA patients are alive and remain free of hematologic disease.Conclusion) FA patients who received AD-HSCT with fludarabine-based conditioning with in vivo T cell depletion using rabbit ATG showed excellent outcome with a lower rate of infectious toxicities than has been reported with conditioning incorporating alemtuzumab. The incidence of primary graft failure should be monitored in a larger cohort of patients. DisclosuresNo relevant conflicts of interest to declare.

Characterization and Genotype-Phenotype Correlation of Fanconi Anemia Patients in Israel

Background: Fanconi Anemia (FA) is an inherited bone marrow failure syndrome in which defects in the DNA repair pathway lead to chromosomal instability. The clinical phenotype includes multiple congenital anomalies, aplastic anemia, an increased tendency to develop malignancies, and extreme sensitivity to alkylating agents. To date, mutations in 21 different genes have been detected in FA patients. A clear genotype-phenotype correlation has not been established, with the exception of patients with FANCD1 (BRCA2) and FANCN mutations, in which early onset of malignancy is almost invariably present. One study found that patients with FANCG mutations have a higher risk for leukemia (Faivre et al., 2000), but this was not confirmed in the International Fanconi Anemia Registry (Kutler et al., 2003). Other evidence suggests that the type of mutation correlates better with the phenotype than the specific gene. For example, one report found that patients with FANCA null mutations present with a more severe phenotype than other types of FANCA mutations (Faivre at al., 2000). Conversely, in a separate report, no functional or clinical difference was found between null and missense mutations (Castella et al., 2011). It is possible that specific mutations best correlate with the phenotype (reviewed in Neveling et al., 2009). However, even for a specific mutation, there is a variable phenotypic severity in different ethnicities and among siblings, suggesting a role for gene modifiers and environmental factors.Objective: To gain insight into the natural history of FA patients in Israel and shed light on genotype-phenotype correlations in this population.Methods: Patients with FA were registered by their treating physicians as part of the Israeli inherited bone marrow failure registry (I-IBMFR). Follow-up surveys were performed every year. For all patients for whom DNA was available, a genetic analysis was performed.Results: One hundred and eleven (111) FA patients diagnosed between 1980 and 2016 were registered to the I-IBMFR and followed for a median of 9.7 years. Analysis showed a slight male predominance (53% males, 47% females) and a near-even split between those of Jewish descent (42%) and those of Muslim descent (38%) with a smaller representation of Bedouins (11%), Druze (6%) and Christians (2%). Over 60% of the patients were offspring of consanguineous parents. Overall, 83% of the patients had confirmed mutations in genes encoding DNA repair proteins, the majority detected in the FANCA gene (67%) with the rest in FANCC (13%), FANCG (14%), FANCJ (3%) and FANCD1 (2%). Only 2 patients who underwent genetic analysis remained undiagnosed.Of the 111 patients, 90% had at least one congenital anomaly. No significant association was found between a specific congenital anomaly and the affected Fanconi gene or the type of mutation. Nearly one third of the patients developed myelodysplastic syndrome (MDS), leukemia or a solid tumor. The prevalence of MDS was higher in FANCC patients and lower in FANCG patients, reaching statistical significance (p=0.0078). There was a trend towards a higher prevalence of cancer in FANCD1 patients.Over half of the FA patients in the registry underwent a hematopoietic stem cell transplant (HSCT), without any correlation to the genotype; 64% of the transplanted patients are alive, while the rest died of transplant-related complications or malignancy. Of the 10 patients who developed solid tumors; 4 were post-HSCT with a solid tumor diagnosed at a range of 2.5-18 years post-transplant. Fifty six percent (56%) of the patients who did not undergo a transplant are alive; mortality was mostly due to malignancy or sepsis. Currently, 68 FA patients from the registry (61%) are alive, with a median age of 17 years.Conclusion: We hereby present a large cohort of FA patients in Israel, unique due to ethnic diversity combined with a high degree of consanguinity, the majority genetically diagnosed. Genotypic and clinical features of FA patients from the I-IBMFR correlate well with trends seen in larger previously published FA registries but also bring to light possible new links. Interestingly, MDS was more prevalent in FANCC patients and less in FANCG patients; a finding that will need further confirmation in future studies. Continuation of this registry may help to unravel further genotype-phenotype trends and enhance our understanding of the disease. DisclosuresNo relevant conflicts of interest to declare.

TGF-β1 Production By Bone Marrow Mesenchymal Stem Cells of FANCD2 Differs from Other Fanconi Anemia Complementation Groups

TGF-β secretion from cells in the bone marrow (BM) niche affects hematopoietic stem cell (HSC) fate and has a cardinal role in HSC quiescence. As a component of the BM niche, BM- mesenchymal stem cells (BM-MSCs) may produce abnormal levels of TGF-β in Fanconi anemia (FA) as obtained for HSCs previously and may play a role in bone marrow failure.Herein, we present the molecular and cellular characterization of FA BM-MSCs by addressing their immunophenotypes, proliferation- and differentiation capacities, as well as expression and secretion levels of transforming growth factor beta (TGF-β) isoforms. Proliferative potency of FA cells was determined between P3 to P7 and compared with healthy donors by calculating cumulative population doubling (cPD) at each passage. Senescence of FA patient BM-MSCs was evaluated at P3 and P6 by SA-β-gal activity and compared with donor BM-MSCs. Diepoxybutane (DEB) effect on TGF-β1 expression of BM-MSCs from FA patients and donors was also assessed.In ten FA patients, mutations were classified as FANCA (n=7), FANCG (n=1) and FANCD2 (n=2). Of six were novel mutations, five in FANCA and one in FANCD2 . BM-MSCs immunophenotype and differentiation capacity of FA BM-MSCs were similar to healthy donors.The CD200 (OX-2) levels were slightly lower in FA MSCs compared to donors. The percentage of CD106 (VCAM-1) positive cells were slightly high especially in FANCD2 cells. FA BM-MSCs showed arrest in dip G2 following DEB treatment. BM-MSCs derived from FANCA- or FANCG -deficient patients displayed similar ex vivo proliferative capacity as healthy donor cells from P3 to P7. However, FANCD2-deficient cells presented lower proliferative capacity. FANCA BM-MSCs had notably increased β-gal positivity at P6, while cells displaying FANCD2 mutation had higher activity at P3 indicating premature aging of these cells. Relative gene expression of TGF-β1 was similar between donor and FA MSCs and did not change by DEB treatment. All cells did not secrete TGF-β2 and -β3. Since the variation of TGF-β1 secretion by FANCA and donor cells was high, a significant difference between the groups could not be obtained. On the other hand, FANCD2 -deficient cells were lack of TGF-β1 secretion. Our data suggest that absence of TGF-β1 secretion may lead to early onset of senescence of the FA-D2 BM-MSCs and may be of value to understand the Fanconi anaemia bone marrow dysfunction in humans. DisclosuresNo relevant conflicts of interest to declare.

Lnk/Sh2b3 Deficiency Restores Hematopoietic Stem Cell Function and Genome Integrity in Fanconi Anemia

▪Although initially identified over 85 years ago, Fanconi Anemia (FA) remains a fatal genetic disease. Patients with FA exhibit developmental malformations, bone marrow failure, and increased cancer susceptibility. Twenty-one FA genes cooperate in a genome stability pathway that is essential for repair of DNA interstrand crosslinks (ICLs) and attenuation of replication stress. Cells derived from FA patients are hypersensitive to ICL-inducing agents such as Mitomycin C (MMC), a hallmark of FA. In addition, FA hematopoietic stem and progenitor cells (HSPCs) show growth retardation and increased apoptosis culminating in hematopoietic stem cell (HSC) exhaustion and bone marrow failure. The poor growth of HSPCs also poses a significant obstacle in gene therapy for FA patients. Moreover, unrepaired DNA damage increases genome instability and promotes leukemia and cancer. Of note, interventions to mitigate HSPC defects in FA do not exist, aside from allogeneic bone marrow transplantation.Remarkably, we reveal here that loss of the adaptor protein Lnk (also called Sh2b3) suppresses FA associated phenotypes without further accelerating neoplastic transformation. LNK is a negative regulator of HSC self-renewal in part by attenuating cytokine signaling, in particular thrombopoietin (TPO)-activated JAK2 signaling in HSCs. Fancd2-/-;Lnk-/- mice exhibit increased phenotypic HSCs in comparison to wildtype (WT) animals. The reconstituting ability of Fancd2-/-;Lnk-/- HSCs is also largely restored to WT levels in serial transplantation assays. In addition, Lnk deficiency restored the growth and survival of FA HSPCs in ex vivo culture. More importantly, Lnk deficiency mitigated chromosomal aberrations associated with FA upon proliferation. To determine if Lnk deficiency mitigates ICL repair, we examined MMC sensitivity in primary HSPCs that were dually null for Lnk and FancD2. Our data suggest that LNK does not play an overt role in ICL repair. Rather, Lnk deficiency in HSPCs mitigated replication stress by stabilizing replication forks and reduces genome instability associated with FA. Hence, our data suggest that Lnk deficiency alleviates replication stress-induced DNA damage and genome instability, thereby restoring cell proliferation and survival of Fancd2-/- HSPCs ex vivo and HSC functions in vivo.Last and importantly, we began to test if LNK inhibition restores human FA HSPCs. We first generated FA-like HSPCs by knocking down FANCD2 in normal umbilical cord blood (UCB) CD34+ cells. Our results demonstrated that shRNA-mediated depletion of LNK in human CD34+ cells rescued progenitor cell numbers and growth in FA-like HSPCs. Take together, our findings reveal a previously unappreciated role of cytokine/JAK signaling in alleviating replication stress and promoting replication-associated genome maintenance. Our studies also illuminate replication stress as a major underlying origin of bone marrow failure in FA patients and have implications for therapeutic strategies to treat FA associated bone marrow failure. DisclosuresNo relevant conflicts of interest to declare.

Re-Analysis of Next Generation Sequencing Gene Panel Data By Normalized Coverage Values Reveals Previously Undetected Copy Number Variations in Inherited Bone Marrow Failure Syndromes

Background: Inherited bone marrow failure syndromes (IBMFSs) comprise a group of genetic disorders affecting blood cell production in bone marrow, resulting in single or multilineage cytopenias. Patients with IBMFSs often have physical malformations and are at risk of developing leukemia and solid tumors. With advances in sequencing technologies, causal nucleotide-level mutations can readily be identified in many IBMFSs. In about half of IBMFS cases, however, a molecular mutation is not detected. Copy number variation (CNVs) have been reported to cause IBMFSs. Unfortunately, genome-wide methods to identify CNVs have major limitations as they may miss small lesions (e.g. by oligonucleotide or CGH arrays) or may have low sensitivity due to low read depths (e.g. by whole genome sequencing).Purpose: This study's purpose was to determine, after standard analysis of a next generation sequencing gene panel for causal mutations, whether re-analysis by normalized coverage value could identified CNVs and to characterize the genetic lesions.Methods & Materials: Patients with IBMFSs were enrolled in The Canadian Inherited Marrow Failure Registry (CIMFR). Genomic DNA was analyzed by next-generation panel gene panel of over 72 IBMFS-associated genes. After analysis for nucleotide level mutations, the NextGene software was used to detect copy number variation using nucleotide read depth in test samples versus normalized control coverage values. Monoalleic copy number deletions were indicated by ratio of <0.33;biallelic deletion was considered when the value was close to zero. PCR or Affymetrix SNP6.0 array were used to validate deletions.Results: Of the 258 patients who tested by the IBMFS next-generation gene panel, 93 (36%) were found to have causal nucleotide level mutations. 168 patients without identified mutations underwent analysis for copy number variation, of which 10 (6%) were found to have deletions. Genes most commonly exhibiting monoallelic deletions included RPS19, RPL11 and RPL5 (5 Diamond-Blackfan anemia patients). One patients with MDS was found to have monoalleic deletion of GATA2 . Moreover, a bialleic deletion of exon 1-5 in FANCA gene was detected in one Fanconi anemia patient. Lastly, we also identified monoalleic deletions of RBM8A in two thrombocytopenia with absent radii patients and of PARN in one unclassified pancytopenia patient. The last three patients were found to also have a nucleotide level mutation on the other allele by the NGS panel (compound heterozygousity). All the above deletions were validated by another method (PCR or Affymetrix SNP6.0 array).Conclusions: CNVs can be detected in some IBMFSs by next-generation sequencing (NGS) panels by careful analysis of normalized coverage values. Such analysis should become standard practice before usage of other mutation detection strategies such as CNV microarray or whole exome/genome sequencing, since the later techniques may not detect certain CNVs that can be detected by gene panels. DisclosuresKlaassen:Amgen: Consultancy; Baxalta: Honoraria; Biogen Canada LTD: Consultancy; Hoffman-La Roche LTD: Consultancy; Agios Pharmaceuticals: Consultancy; Octapharma: Honoraria. Dror:Alexion Canada: Research Funding.

Clonal Cytogenetic Abnormalities in Myeloid Neoplasms Arising from Fanconi Anemia — a Single Center Study from India

Fanconi anemia (FA) is a genomic instability disease caused by defective DNA repair which predisposes patients into developing myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Identification of clonal abnormalities in FA patients is vital to detect transformation before the onset of a high-grade MDS/leukemia.We analyzed bone marrow (BM) samples from a series of 50 patients with FA who were diagnosed at the Christian Medical College, Vellore, India from January 2005 - July 2017 by peripheral chromosome breakage analysis (CBA) or FANCD2 ubiquitination analysis of peripheral blood or dermal fibroblasts. Inclusion in the current study was based on the availability of karyotyping samples performed on the BM aspirate collected at diagnosis. Peripheral blood and dermal fibroblasts were collected at diagnosis for CBA and FANCD2 western blot.All 50 patients evaluated had phenotypic features suggestive of FA. There were 28 patients who had increased CBA scores alone while 21 had both increased CBA scores as well as defective FANCD2 ubiquitination. One patient with negative CBA score showing hematopoietic mosaicism with defective FANCD2 ubiquitination pattern in dermal fibroblasts alone was also included in the study.The median age at diagnosis was 16 years (range 1-35) and 35 (70%) were male. Abnormal karyotypes were obtained in 38 patients while the remaining (n=12) showed a normal karyotype. Examination of bone marrow aspirate and trephine was consistent with MDS in 29 (MDS-RS-MLD n= 3; MDS-EB n= 10; MDS-U n=12; h-MDS n=4), while the remaining had either AML (n=8) or a hypoplastic marrow (n=13). The 12 patients who had a normal karyotype were predominantly hypoplastic (n=8) or had either MDS (n=3) or AML (n=1). Abnormalities of chromosomes 1 and 7 were the most commonly reported chromosome aberration and was seen in 17 patients each (45%). A partial deletion 5q was present in 7 (18%) patients while gains of chromosomes 8 and 21 were seen in 5 and 6 patients respectively. Complex karyotypes involving abnormalities of 3 or more chromosomes were seen in 16 (42%) patients who had MDS/AML. FA gene mutation analysis of 8 patients showed FANCA gene mutations in 7 patients (4-abnormal KT, 3-normal KT) while 1 patient with abnormal karyotype had a mutation in the FANCJ gene.Post treatment karyotypes were available in 2 patients with AML and 1 patient with MDS which showed persistence of the leukemic clone identified at diagnosis (n=1) or clonal evolution (n=1, gain of chromosome 9) while the third patient showed a new clone after treatment which retained two of the three abnormalities documented at diagnosis.Using the Revised International Prognostic Scoring system for MDS majority of the patients (n=20) with MDS were classified in the poor or very poor risk groups while the remaining patients were in the good (n=6) or intermediate (n=3) risk group.Five of the 8 patients with AML were classified as poor risk according to the refined MRC cytogenetic classification while 3 were grouped in the intermediate risk group.Among the 13 patients showing a hypoplastic marrow, one patient had monosomy 7 which has been shown to have an increased risk of transformation to MDS/AML. One patient who had gain of 1q due to an unbalanced translocation transformed to AML in 9 months.We report a distinct pattern of chromosome abnormalities in patients with FA-MDS/AML consistent with previous reports, underscoring the need for inclusion of karyotype analysis in the laboratory evaluation strategy of FA patients for early detection of transformation to hematologic malignancies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Comparable Clinical Outcomes between Haploidentical Family Donor and Other Donor Types after Hematopoietic Stem Cell Transplantation for Rare Pediatric Rare non-Malignant Diseases

Introduction : Hematopoietic stem cell transplantation (HSCT) has been applied as the curative treatment for rare pediatric non-malignant diseases. However, these patients are at risk of transplant related morbidity and there are no established conditioning regimens, especially for children without matched sibling donors. We evaluate the outcome of 43 pediatric cases with rare non-malignant diseases treated with HSCT.Method : We performed a retrospective analysis of a total of 43 transplantations performed in 41 patients with rare non-malignant diseases between May 2002 and May 2017. The conditioning regimens were myeloablative (busulfan, cyclophosphamide) until the middle of 2011, and reduce intensity regimens were applied thereafter. Fanconi anemia patients received fludarabine, cyclophosphamide with or without ATG. In haploidentical hematopoietic stem cell transplantation (HHCT) before year 2016 a combination of fludarabine, cyclophosphamide and ATG was mostly used, while uniformed combination of fludarabine, thiotepa, treosulfan and ATG was used since 2016. Grafts for HHSCT were manipulated by ex vivo CD3 or αβ+ T-cell depletion.Results : This study included 14 patients with congenital immunodeficiency, 13 with congenital bone marrow failure syndrome, 5 with degenerative metabolic disease and 7 with familial hemophagocytic lymphohiotiocytosis (Table 1). By donor types, 8 patients received grafts from matched familial donor (MFD), 22 from matched unrelated donor (MUD), and 10 from haploidentical family donor(HFD), respectively.The median age at HSCT was 2.5 years (range, 0.65-13.5 years). Grafts for HHCTs were CD3-depleted in 2 patients and αβ+ T-cell depleted in 8. The median infused dose of CD34+ cells were 9.38 x 106/kg (range, 0.15-44.11). Among 41 patients, 39 patients reached neutrophil engraftment, with a median of 11 days (range, 9-21) with no significant difference in neutrophil engraftment between donor types (P value 0.355).Graft failure (GF) occurred in 4 patients (primary in 3 patients; Omenn syndrome, Kostmann syndrome, Thalassemia and secondary in 1 patient; Wiskott-Aldrich syndrome). One expired due to sepsis, and 1 patient diagnosed with thalassemia is alive with occasional red blood cell transfusion on an outpatient basis. The other two underwent second HHCT; one with secondary GF died due to pneumonia after engraftment, and the other experienced GF but alive with supportive care.Mixed chimerism was detected in 24 patients 1 month after HSCT (range, 1 - 100%) and in 10 patients 1 year after HSCT (range, 1 - 80%). Grade II-IV acute GVHD occurred in 12 patients (29%, 5 with Gr II, 5 with Gr III and 2 with Gr IV), while chronic GVHD occurred in 4 patients (10%, 1 MFD, 3 MUD and all extensive). There were 4 patients who developed cytomegalovirus (CMV) disease, and 2 with sinusoidal obstruction syndrome (SOS), but no post-transplant lymphoproliferative disorder. There was no mortality related to GVHD, CMV or SOS.As for survival results, a total of 6 patients died; one HFD recipient died of pneumonia on day +216 of second HHCT, 3 MUD recipients died of pulmonary hemorrhage and the other 1 MUD recipient died of sepsis. One MUD patient died due to sudden cardiac arrest 3 years after HSCT. The overall transplant-related mortality was 14.6%. At a median follow up 56 months (range, 0.3 - 182 months), the 5-year overall survival (OS) and event free survival (EFS) was 85.4% and 82.9%, respectively. There was no statistical difference in OS and EFS among HSCTs from 3 different donor types (OS - MFD : 100% vs MUD : 72.7% vs HFD : 90.0%, P=0.336, EFS-MFD : 100% vs MUD : 72.7% vs HFD : 80.0%, P=0.367). Patients who received HSCT at younger age (<500 days) had a worse outcome compared to the older age group (OS : 54.5% vs 96.7%, P=0.001, EFS : 63.6% vs 90.0%, P=0.038). Patients in the congenital immunodeficiency group tended to have a lower OS, although this showed no statistically significant difference (P=0.074).Conclusion : In this study, we presented a single center experience of HSCT in pediatric rare non-malignant diseases and showed that HHCT donors were comparable to HSCT from other donor types, with no statistically significant difference in neutrophil engraftment, OS and EFS. Since an HLA haploidentical relative is available in almost every patient, the improvement of this treatment modality will offer a better chance of cure in patients with a variety of pediatric rare non-malignant diseases. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Novel Lineage Depletion and Manufacturing Allows for Unprecedented Preservation of Autologous Blood Stem Cells for Gene Therapy of Fanconi Anemia Complementation Group Α

A hallmark of the rare, monogenic disorder called Fanconi anemia (FA) is an accelerated decline in hematopoietic stem cells (HSC) leading to bone marrow (BM) failure. Long-term treatment requires hematopoietic cell transplantation (HCT) from an unaffected donor, but success of this approach in ~70% of the patients lacking a matched sibling is associated with potentially severe side effects. Gene therapy could be an alternative, correcting the genetic defect in the patient's own HSC and negating the need for a HCT donor. Building on previous FA gene therapy studies, we developed an optimized protocol for lentivirus (LV)-mediated FANCA gene transfer into HSCs from FA-A patients [Becker, P. Human Gene Therapy, 2010]. This phase I clinical trial incorporates vector recommendations from the International FA Gene Therapy Working Group. Three patients have been treated on this trial to date (National Clinical Trials Registry ID: NCT01331018). This protocol collects unmanipulated HSC from BM or mobilized leukapheresis (mAPH) and does not include conditioning prior to cell infusion. In the first two patients enrolled, harvested BM demonstrated reduced CD34+ cell numbers and CD34 expression (Figure 1). In the first patient, direct enrichment of dimly CD34+ cells was very inefficient. Thus, for the second patient the enrichment step was eliminated. While this processing change preserved more of the CD34+ cells and improved transduction and viability, it required a prohibitive amount of LV vector. To reduce vector needs and preserve available CD34+ cells for future FA patients, we developed a novel strategy to deplete lineage+ (CD3+, CD14+, CD16+ and CD19+) cells from both mAPH and BM products. This process efficiently depletes >90% of lineage+ cells from healthy donor cell products while retaining ≥60% of the initial CD34+ cell fraction, reduced total nucleated cells (TNC) by 1-2 logs and maintained improved transduction and viability following gene transfer. Most importantly, transduced lineage- cell products engrafted in an immune deficient mouse model equivalent to that of purified CD34+ cells from the same donor when transplanted at matched CD34+ cell doses. This novel selection strategy has been approved by the regulatory agencies and a third patient was treated in July 2017 with this approach. Patient 3 is a 5-year old male with confirmed FA-A by complementation studies. Baseline ANC averaged 1.6 K/mcL and baseline platelet counts averaged 31 K/mcL in the 6 months prior to treatment, with declining ANC and platelets over a 4 year interval prior to gene therapy. Successful mobilization of CD34+ cells was achieved with combination filgrastim (16µg/kg BID or 32µg/kg/day) and plerixafor (240µg/kg). Two, successive apheresis collections resulted in 8.5×1010 TNC containing 1.6×108 CD34+ cells. A total of 5×1010 TNC were subjected to lineage depletion, resulting in a 94% reduction in TNC and a 55% retention of available CD34+ cells. A total of 52.4×106 CD34+ cells were transduced at 5 IU/cell, resulting in a final cell dose of 2.4×106 CD34+ cells per kg. We observed a VCN of 1.8 copies per cell and viability of the final cellular product was 99.3%. Infusion was well-tolerated and a fourth patient is expected to be treated in the Fall of 2017. This novel method of depleting lineage positive cells reduced TNC counts to feasible numbers for transduction while maintaining high numbers of CD34+ cells, a strategy which could have benefits in other disease targets for gene therapy. [Display omitted] DisclosuresAdair:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties, Research Funding; CalImmune Inc.: Consultancy. Becker:GlycoMimetics, Inc.: Research Funding. Kiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.