T-Cell Lymphomas in Recipients of CAR-T Cells: Assessing Risks and Causalities.

The United States Food and Drug Administration (FDA) announcement in November 2023 regarding reports of the occurrence of secondary T-cell lymphomas in patients receiving chimeric antigen receptor T-cells (CAR-T) for B-cell malignancies resulted in widespread concern among patients, clinicians and scientists. Little information relevant to assessing causality, most importantly whether CAR retroviral or lentiviral vector genomic insertions contribute to oncogenesis, was initially available. However, since that time several publications have provided clinical and molecular details on three cases showing clonal CAR vector insertions in tumor cells but without firm evidence these insertions played any role in oncogenic transformation. In addition, several other cases have been reported without vector detected in tumor cells. In addition, epidemiologic analyses as well as institutional long-term CAR-T recipient cohort studies provide important additional information suggesting the risk of T-cell lymphomas following CAR-T therapies is extremely low. This review will provide a summary of information available to date, as well as review relevant prior research suggesting a low susceptibility of mature T-cells to insertional oncogenesis and documenting the almost complete lack of T-cell transformation following natural HIV infection. Alternative factors that may predispose patients treated with CAR-T cells to secondary hematologic malignancies, including immune dysfunction and clonal hematopoiesis, are discussed and likely play a greater role than insertional mutagenesis in secondary malignancies post-CAR therapies.

Clonal landscape and clinical outcomes of telomere biology disorders: somatic rescuing and cancer mutations.

Telomere biology disorders (TBD), caused by pathogenic germline variants in telomere-related genes, present with multi-organ disease and a predisposition to cancer. Clonal hematopoiesis (CH) as a marker of cancer development and survival in TBD is poorly understood. Here, we characterized the clonal landscape of a large cohort of 207 TBD patients with a broad range of age and phenotype. CH occurred predominantly in symptomatic patients and in signature genes typically associated with cancers: PPM1D, POT1, TERT promoter (TERTp), U2AF1S34, and/or TP53. Chromosome 1q gain (Chr1q+) was the commonest karyotypic abnormality. Clinically, multiorgan involvement and CH in TERTp, TP53, and splicing factor genes associated with poorer overall survival. Chr1q+, and splicing factor or TP53 mutations significantly increased the risk of hematologic malignancies, regardless of the clonal burden. Chr1q+ and U2AF1S34 mutated clones were pre-malignant events associated with the secondary acquisition of mutations in genes related to hematologic malignancies. Like known effects of Chr1q+ and TP53-CH, functional studies demonstrated that U2AF1S34 mutations primarily compensated for aberrant upregulation of TP53 and interferon pathways in telomere-dysfunctional hematopoietic stem cells, highlighting the TP53 pathway as a canonical route of malignancy in TBD. In contrast, somatic POT1/PPM1D/TERTp-CH had distinct trajectories unrelated to cancer development. With implications beyond TBD, our data show that telomere dysfunction is a strong selective pressure for CH. In TBD, CH is a poor prognostic marker associated with worse overall survival. The identification of key regulatory pathways that drive clonal transformation in TBD allows the identification of patients at a higher risk of cancer development.

Long-term engraftment and maturation of autologous iPSC-derived cardiomyocytes in two rhesus macaques.

Cellular therapies with cardiomyocytes produced from induced pluripotent stem cells (iPSC-CMs) offer a potential route to cardiac regeneration as a treatment for chronic ischemic heart disease. Here, we report successful long-term engraftment and in vivo maturation of autologous iPSC-CMs in two rhesus macaques with small, subclinical chronic myocardial infarctions, all without immunosuppression. Longitudinal positron emission tomography imaging using the sodium/iodide symporter (NIS) reporter gene revealed stable grafts for over 6 and 12 months, with no teratoma formation. Histological analyses suggested capability of the transplanted iPSC-CMs to mature and integrate with endogenous myocardium, with no sign of immune cell infiltration or rejection. By contrast, allogeneic iPSC-CMs were rejected within 8 weeks of transplantation. This study provides the longest-term safety and maturation data to date in any large animal model, addresses concerns regarding neoantigen immunoreactivity of autologous iPSC therapies, and suggests that autologous iPSC-CMs would similarly engraft and mature in human hearts.

In vivo tracking of ex vivo generated 89 Zr-oxine labeled plasma cells by PET in a non-human primate model.

B cells are an attractive platform for engineering to produce protein-based biologics absent in genetic disorders, and potentially for the treatment of metabolic diseases and cancer. As part of pre-clinical development of B cell medicines, we demonstrate a method to collect, ex vivo expand, differentiate, radioactively label, and track adoptively transferred non-human primate (NHP) B cells. These cells underwent 10- to 15-fold expansion, initiated IgG class switching, and differentiated into antibody secreting cells. Zirconium-89-oxine labeled cells were infused into autologous donors without any preconditioning and tracked by PET/CT imaging. Within 24 hours of infusion, 20% of the initial dose homed to the bone marrow and spleen and distributed stably and equally between the two. Interestingly, approximately half of the dose homed to the liver. Image analysis of the bone marrow demonstrated inhomogeneous distribution of the cells. The subjects experienced no clinically significant side effects or laboratory abnormalities. A second infusion of B cells into one of the subjects resulted in an almost identical distribution of cells, suggesting a non-limiting engraftment niche and feasibility of repeated infusions. This work supports the NHP as a valuable model to assess the potential of B cell medicines as potential treatment for human diseases.

An epigenetically distinct HSC subset supports thymic reconstitution.

Hematopoietic stem cells (HSCs) with multilineage potential are critical for effective T cell reconstitution and restoration of the adaptive immune system after allogeneic Hematopoietic Cell Transplantation (allo-HCT). The Kit lo subset of HSCs is enriched for multipotential precursors, 1, 2 but their T-cell lineage potential has not been well-characterized. We therefore studied the thymic reconstituting and T-cell potential of Kit lo HSCs. Using a preclinical allo-HCT model, we demonstrate that Kit lo HSCs support better thymic recovery, and T-cell reconstitution resulting in improved T cell responses to infection post-HCT. Furthermore, Kit lo HSCs with augmented BM lymphopoiesis mitigate age-associated thymic alterations, thus enhancing T-cell recovery in middle-aged hosts. We find the frequency of the Kit lo subset declines with age, providing one explanation for the reduced frequency of T-competent HSCs and reduced T-lymphopoietic potential in BM precursors of aged mice. 3, 4, 5 Chromatin profiling revealed that Kit lo HSCs exhibit higher activity of lymphoid-specifying transcription factors (TFs), including Zbtb1 . Deletion of Zbtb1 in Kit lo HSCs diminished their T-cell potential, while reinstating Zbtb1 in megakaryocytic-biased Kit hi HSCs rescued T-cell potential, in vitro and in vivo . Finally, we discover an analogous Kit lo HSC subset with enhanced lymphoid potential in human bone marrow. Our results demonstrate that Kit lo HSCs with enhanced lymphoid potential have a distinct underlying epigenetic program.

Cigarette smoke impairs the hematopoietic supportive property of mesenchymal stem cells via the production of reactive oxygen species and NLRP3 activation.

BACKGROUND: Mesenchymal stem cells (MSCs) play important roles in tissue homeostasis by providing a supportive microenvironmental niche for the hematopoietic system. Cigarette smoking induces systemic abnormalities, including an impeded recovery process after hematopoietic stem cell transplantation. However, the role of cigarette smoking-mediated alterations in MSC niche function have not been investigated. METHODS: In the present study, we investigated whether exposure to cigarette smoking extract (CSE) disrupts the hematopoietic niche function of MSCs, and pathways impacted. To investigate the effects on bone marrow (BM)-derived MSCs and support of hematopoietic stem and progenitor cells (HSPCs), mice were repeatedly infused with the CSE named 3R4F, and hematopoietic stem and progenitor cells (HSPCs) supporting function was determined. The impact of 3R4F on MSCs at cellular level were screened by bulk-RNA sequencing and subsequently validated through qRT-PCR. Specific inhibitors were treated to verify the ROS or NLRP3-specific effects, and the cells were then transplanted into the animal model or subjected to coculture with HSPCs. RESULTS: Both direct ex vivo and systemic in vivo MSC exposure to 3R4F resulted in impaired engraftment in a humanized mouse model. Furthermore, transcriptomic profile analysis showed significantly upregulated signaling pathways related to reactive oxygen species (ROS), inflammation, and aging in 3R4F-treated MSCs. Notably, ingenuity pathway analysis revealed the activation of NLRP3 inflammasome signaling pathway in 3R4F-treated MSCs, and pretreatment with the NLRP3 inhibitor MCC950 rescued the HSPC-supporting ability of 3R4F-treated MSCs. CONCLUSION: In conclusion, these findings indicate that exposure to CSE reduces HSPCs supportive function of MSCs by inducing robust ROS production and subsequent NLRP3 activation.

Clonal hematopoiesis in people with advanced HIV and associated inflammatory syndromes.

People with HIV (PWH) have a higher age-adjusted mortality due to chronic immune activation and age-related comorbidities. PWH also have higher rates of clonal hematopoiesis (CH) than age-matched non-HIV cohorts; however, risk factors influencing the development and expansion of CH in PWH remain incompletely explored. We investigated the relationship between CH, immune biomarkers, and HIV-associated risk factors (CD4+ and CD8+ T cells, nadir CD4+ count, opportunistic infections [OIs], and immune reconstitution inflammatory syndrome [IRIS]) in a diverse cohort of 197 PWH with median age of 42 years, using a 56-gene panel. Seventy-nine percent had a CD4+ nadir below 200 cells/µL, 58.9% had prior OIs, and 34.5% had a history of IRIS. The prevalence of CH was high (27.4%), even in younger individuals, and CD8+ T cells and nadir CD4+ counts strongly associated with CH after controlling for age. A history of IRIS was associated with CH in a subgroup analysis of patients 35 years of age and older. Inflammatory biomarkers were higher in CH carriers compared with noncarriers, supporting a dysregulated immune state. These findings suggest PWH with low nadir CD4+ and/or inflammatory complications may be at high risk of CH regardless of age and represent a high-risk group that could benefit from risk reduction and potentially targeted immunomodulation.

Telomerase is essential for cardiac differentiation and sustained metabolism of human cardiomyocytes.

Telomeres as the protective ends of linear chromosomes, are synthesized by the enzyme telomerase (TERT). Critically short telomeres essentially contribute to aging-related diseases and are associated with a broad spectrum of disorders known as telomeropathies. In cardiomyocytes, telomere length is strongly correlated with cardiomyopathies but it remains ambiguous whether short telomeres are the cause or the result of the disease. In this study, we employed an inducible CRISPRi human induced pluripotent stem cell (hiPSC) line to silence TERT expression enabling the generation of hiPSCs and hiPSC-derived cardiomyocytes with long and short telomeres. Reduced telomerase activity and shorter telomere lengths of hiPSCs induced global transcriptomic changes associated with cardiac developmental pathways. Consequently, the differentiation potential towards cardiomyocytes was strongly impaired and single cell RNA sequencing revealed a shift towards a more smooth muscle cell like identity in the cells with the shortest telomeres. Poor cardiomyocyte function and increased sensitivity to stress directly correlated with the extent of telomere shortening. Collectively our data demonstrates a TERT dependent cardiomyogenic differentiation defect, highlighting the CRISPRi TERT hiPSCs model as a powerful platform to study the mechanisms and consequences of short telomeres in the heart and also in the context of telomeropathies.

Treatment of refractory/relapsed Diamond-Blackfan anaemia with eltrombopag.

Diamond-Blackfan anaemia (DBA) is a rare, inherited bone marrow failure syndrome with a ribosomal defect causing slowed globin chain production with normal haem synthesis, causing an overabundance of reactive iron/haem and erythroid-specific cellular toxicity. Eltrombopag, a non-peptide thrombopoietin receptor agonist, is a potent intracellular iron chelator and induced a robust durable response in an RPS19-mutated DBA patient on another trial. We hypothesized eltrombopag would improve RBC production in DBA patients. We conducted a single-centre, single-arm pilot study (NCT04269889) assessing safety and erythroid response of 6 months of daily, fixed-dose eltrombopag for DBA patients. Fifteen transfusion-dependent (every 3-5 weeks) patients (median age 18 [range 2-56]) were treated. One responder had sustained haemoglobin improvement and >50% reduction in RBC transfusion frequency. Of note, 7/15 (41%) patients required dose reductions or sustained discontinuation of eltrombopag due to asymptomatic thrombocytosis. Despite the low response rate, eltrombopag has now improved erythropoiesis in several patients with DBA with a favourable safety profile. Dosing restrictions due to thrombocytosis may cause insufficient iron chelation to decrease haem production and improve anaemia in most patients. Future work will focus on erythropoiesis dynamics in patients and use of haem synthesis inhibitors without an impact on other haematopoietic lineages.

Impact of CRISPR/HDR editing versus lentiviral transduction on long-term engraftment and clonal dynamics of HSPCs in rhesus macaques.

For precise genome editing via CRISPR/homology-directed repair (HDR), effective and safe editing of long-term engrafting hematopoietic stem cells (LT-HSCs) is required. The impact of HDR on true LT-HSC clonal dynamics in a relevant large animal model has not been studied. To track the output and clonality of HDR-edited cells and to provide a comparison to lentivirally transduced HSCs in vivo, we developed a competitive rhesus macaque (RM) autologous transplantation model, co-infusing HSCs transduced with a barcoded GFP-expressing lentiviral vector (LV) and HDR edited at the CD33 locus. CRISPR/HDR-edited cells showed a two-log decrease by 2 months following transplantation, with little improvement via p53 inhibition, in comparison to minimal loss of LV-transduced cells long term. HDR long-term clonality was oligoclonal in contrast to highly polyclonal LV-transduced HSCs. These results suggest marked clinically relevant differences in the impact of current genetic modification approaches on HSCs.

Ongoing production of tissue-resident macrophages from hematopoietic stem cells in healthy adult macaques.

ABSTRACT: Macrophages orchestrate tissue immunity from the initiation and resolution of antimicrobial immune responses to the repair of damaged tissue. Murine studies demonstrate that tissue-resident macrophages are a heterogenous mixture of yolk sac-derived cells that populate the tissue before birth, and bone marrow-derived replacements recruited in adult tissues at steady-state and in increased numbers in response to tissue damage or infection. How this translates to species that are constantly under immunologic challenge, such as humans, is unknown. To understand the ontogeny and longevity of tissue-resident macrophages in nonhuman primates (NHPs), we use a model of autologous hematopoietic stem progenitor cell (HSPC) transplantation with HSPCs genetically modified to be marked with clonal barcodes, allowing for subsequent analysis of clonal ontogeny. We study the contribution of HSPCs to tissue macrophages, their clonotypic profiles relative to leukocyte subsets in the peripheral blood, and their transcriptomic and epigenetic landscapes. We find that HSPCs contribute to tissue-resident macrophage populations in all anatomic sites studied. Macrophage clonotypic profiles are dynamic and overlap significantly with the clonal hierarchy of contemporaneous peripheral blood monocytes. Epigenetic and transcriptomic landscapes of HSPC-derived macrophages are similar to tissue macrophages isolated from NHPs that did not undergo transplantation. We also use in vivo bromodeoxyuridine infusions to monitor tissue macrophage turnover in NHPs that did not undergo transplantation and find evidence for macrophage turnover at steady state. These data demonstrate that the life span of most tissue-resident macrophages is limited and can be replenished continuously from HSPCs.

Granulocyte transfusions in severe aplastic anemia.

Patients with severe aplastic anemia (SAA) are at high risk for morbidity and mortality due to severe infections. We aimed to characterize the role of granulocyte transfusion (GT) in SAA. Primary outcomes were survival from first GT, including overall survival (OS) at last follow up, survival to discharge, and receipt of HSCT. Secondary outcomes included evaluation of clinical response at 7 and 30 days after GT initiation based on a clinical scoring system incorporating microbiological and radiographic response. Twenty-eight SAA patients underwent 30 GT courses with a per-dose median of 1.28 x 109 granulocyte cells/kilogram (range 0.45-4.52 x 109). OS from initial GT to median last follow up (551 days) was 50%, with 39% (11/28) alive at last follow up. Sixty-four percent (18/28) of all patients survived to hospital discharge. Patients with complete, partial, or stable response at 30 days had significantly improved OS compared to non-responders (p=0.0004). Eighty-six percent (18/21) of patients awaiting HSCT during GT underwent transplant and 62% (13/21) survived to post-HSCT discharge. Sex, type of infection, or percentage of days with absolute neutrophil count > 0.2x109/L during GT course were not predictive of survival (p=0.52, p=0.7, p=0.28). Nine of 28 (32%) patients developed new or increased human leukocyte antigen (HLA) alloimmunization during their GT course. GTs in SAA may impact survival in those with improvement or stabilization of their underlying infection. Alloimmunization can occur and OS in this population remains poor, but GTs may be a useful tool to bridge patients to curative treatment with HSCT.

Fertility-preserving myeloablative conditioning using single-dose CD117 antibody-drug conjugate in a rhesus gene therapy model.

Hematopoietic stem cell (HSC) gene therapy has curative potential; however, its use is limited by the morbidity and mortality associated with current chemotherapy-based conditioning. Targeted conditioning using antibody-drug conjugates (ADC) holds promise for reduced toxicity in HSC gene therapy. Here we test the ability of an antibody-drug conjugate targeting CD117 (CD117-ADC) to enable engraftment in a non-human primate lentiviral gene therapy model of hemoglobinopathies. Following single-dose CD117-ADC, a >99% depletion of bone marrow CD34 + CD90 + CD45RA- cells without lymphocyte reduction is observed, which results are not inferior to multi-day myeloablative busulfan conditioning. CD117-ADC, similarly to busulfan, allows efficient engraftment, gene marking, and vector-derived fetal hemoglobin induction. Importantly, ADC treatment is associated with minimal toxicity, and CD117-ADC-conditioned animals maintain fertility. In contrast, busulfan treatment commonly causes severe toxicities and infertility in humans. Thus, the myeloablative capacity of single-dose CD117-ADC is sufficient for efficient engraftment of gene-modified HSCs while preserving fertility and reducing adverse effects related to toxicity in non-human primates. This targeted conditioning approach thus provides the proof-of-principle to improve risk-benefit ratio in a variety of HSC-based gene therapy products in humans.

Investigation of the impact of clonal hematopoiesis on severity and pathophysiology of COVID-19 in rhesus macaques.

Clinical manifestations of COVID-19 vary widely, ranging from asymptomatic to severe respiratory failure with profound inflammation. Although risk factors for severe illness have been identified, definitive determinants remain elusive. Clonal hematopoiesis (CH), the expansion of hematopoietic stem and progenitor cells bearing acquired somatic mutations, is associated with advanced age and hyperinflammation. Given the similar age range and hyperinflammatory phenotype between frequent CH and severe COVID-19, CH could impact the risk of severe COVID-19. Human cohort studies have attempted to prove this relationship, but conclusions are conflicting. Rhesus macaques (RMs) are being utilized to test vaccines and therapeutics for COVID-19. However, RMs, even other species, have not yet been reported to develop late inflammatory COVID-19 disease. Here, RMs with either spontaneous DNMT3A or engineered TET2 CH along with similarly transplanted and conditioned controls were infected with SARS-CoV-2 and monitored until 12 days post-inoculation (dpi). Although no significant differences in clinical symptoms and blood counts were noted, an aged animal with natural DNMT3A CH died on 10 dpi. CH macaques showed evidence of sustained local inflammatory responses compared to controls. Interestingly, viral loads in respiratory tracts were higher at every timepoint in the CH group. Lung sections from euthanasia showed evidence of mild inflammation in all animals, while viral antigen was more frequently detected in the lung tissues of CH macaques even at the time of autopsy. Despite the lack of striking inflammation and serious illness, our findings suggest potential pathophysiological differences in RMs with or without CH upon SARS-CoV-2 infection.

Arginine metabolism regulates human erythroid differentiation through hypusination of eIF5A.

Metabolic programs contribute to hematopoietic stem and progenitor cell (HSPC) fate, but it is not known whether the metabolic regulation of protein synthesis controls HSPC differentiation. Here, we show that SLC7A1/cationic amino acid transporter 1-dependent arginine uptake and its catabolism to the polyamine spermidine control human erythroid specification of HSPCs via the activation of the eukaryotic translation initiation factor 5A (eIF5A). eIF5A activity is dependent on its hypusination, a posttranslational modification resulting from the conjugation of the aminobutyl moiety of spermidine to lysine. Notably, attenuation of hypusine synthesis in erythroid progenitors, by the inhibition of deoxyhypusine synthase, abrogates erythropoiesis but not myeloid cell differentiation. Proteomic profiling reveals mitochondrial translation to be a critical target of hypusinated eIF5A, and accordingly, progenitors with decreased hypusine activity exhibit diminished oxidative phosphorylation. This affected pathway is critical for eIF5A-regulated erythropoiesis, as interventions augmenting mitochondrial function partially rescue human erythropoiesis under conditions of attenuated hypusination. Levels of mitochondrial ribosomal proteins (RPs) were especially sensitive to the loss of hypusine, and we find that the ineffective erythropoiesis linked to haploinsufficiency of RPS14 in chromosome 5q deletions in myelodysplastic syndrome is associated with a diminished pool of hypusinated eIF5A. Moreover, patients with RPL11-haploinsufficient Diamond-Blackfan anemia as well as CD34+ progenitors with downregulated RPL11 exhibit a markedly decreased hypusination in erythroid progenitors, concomitant with a loss of mitochondrial metabolism. Thus, eIF5A-dependent protein synthesis regulates human erythropoiesis, and our data reveal a novel role for RPs in controlling eIF5A hypusination in HSPCs, synchronizing mitochondrial metabolism with erythroid differentiation.

Investigation of the Impact of Clonal Hematopoiesis on Severity and Pathophysiology of COVID-19 in Rhesus Macaques.

Clinical manifestations of COVID-19 vary widely, ranging from asymptomatic to severe respiratory failure with profound inflammation. Although risk factors for severe illness have been identified, definitive determinants remain elusive. Clonal hematopoiesis (CH), the expansion of hematopoietic stem and progenitor cells bearing acquired somatic mutations, is associated with advanced age and hyperinflammation. Given the similar age range and hyperinflammatory phenotype between frequent CH and severe COVID-19, CH could impact the risk of severe COVID-19. Human cohort studies have attempted to prove this relationship, but conclusions are conflicting. Rhesus macaques (RMs) are being utilized to test vaccines and therapeutics for COVID-19. However, RMs, even other species, have not yet been reported to develop late inflammatory COVID-19 disease. Here, RMs with either spontaneous DNMT3A or engineered TET2 CH along with similarly transplanted and conditioned controls were infected with SARS-CoV-2 and monitored until 12 days post-inoculation (dpi). Although no significant differences in clinical symptoms and blood counts were noted, an aged animal with natural DNMT3A CH died on 10 dpi. CH macaques showed evidence of sustained local inflammatory responses compared to controls. Interestingly, viral loads in respiratory tracts were higher at every timepoint in the CH group. Lung sections from euthanasia showed evidence of mild inflammation in all animals, while viral antigen was more frequently detected in the lung tissues of CH macaques even at the time of autopsy. Despite the lack of striking inflammation and serious illness, our findings suggest potential pathophysiological differences in RMs with or without CH upon SARS-CoV-2 infection. Highlights: No evidence of association between CH and COVID-19 clinical severity in macaques.The presence of CH is associated with prolonged local inflammatory responses in COVID-19.SARS-CoV-2 persists longer in respiratory tracts of macaques with CH following infection.

Expanded NK cells used for adoptive cell therapy maintain diverse clonality and contain long-lived memory-like NK cell populations.

Multiple clinical trials exploring the potential of adoptive natural killer (NK) cell therapy for cancer have employed ex vivo expansion using feeder cells to obtain large numbers of NK cells. We have previously utilized the rhesus macaque model to clonally track the NK cell progeny of barcode-transduced CD34+ stem and progenitor cells after transplant. In this study, NK cells from barcoded rhesus macaques were used to study the changes in NK cell clonal patterns that occurred during ex vivo expansion using culture protocols similar to those employed in clinical preparation of human NK cells including irradiated lymphoblastoid cell line (LCL) feeder cells or K562 cells expressing 4-1BBL and membrane-bound interleukin-21 (IL-21). NK expansion cultures resulted in the proliferation of clonally diverse NK cells, which, at day 14 harvest, contained greater than 50% of the starting barcode repertoire. Diversity as measured by Shannon index was maintained after culture. With both LCL and K562 feeders, proliferation of long-lived putative memory-like NK cell clones was observed, with these clones continuing to constitute a mean of 31% of the total repertoire of expanded cells. These experiments provide insight into the clonal makeup of expanded NK cell clinical products.

Comparison of busulfan and total body irradiation conditioning on hematopoietic clonal dynamics following lentiviral gene transfer in rhesus macaques.

The clonal dynamics following hematopoietic stem progenitor cell (HSPC) transplantation with busulfan conditioning are of great interest to the development of HSPC gene therapies. Compared with total body irradiation (TBI), busulfan is less toxic and more clinically relevant. We used a genetic barcoded HSPC autologous transplantation model to investigate the impact of busulfan conditioning on hematopoietic reconstitution in rhesus macaques. Two animals received lower busulfan dose and demonstrated lower vector marking levels compared with the third animal given a higher busulfan dose, despite similar busulfan pharmacokinetic analysis. We observed uni-lineage clonal engraftment at 1 month post-transplant, replaced by multilineage clones by 2 to 3 months in all animals. The initial multilineage clones in the first two animals were replaced by a second multilineage wave at 9 months; this clonal pattern disappeared at 13 months in the first animal, though was maintained in the second animal. The third animal maintained stable multilineage clones from 3 months to the most recent time point. In addition, busulfan animals exhibit more rapid HSPC clonal mixing across bone marrow sites and less CD16+ NK-biased clonal expansion compared with TBI animals. Therefore, busulfan conditioning regimens can variably impact the marrow niche, resulting in differences in clonal patterns with implications for HSPC gene therapies.