High-level protein production in erythroid cells derived from in vivo transduced hematopoietic stem cells

Wang, Hongjie; Liu, Zhinan; Li, Chang; Gil, Sucheol; Papayannopoulou, Thalia; Doering, Christopher B.; Lieber, André

doi:10.1182/bloodadvances.2019000706

Cited by 20 publications

(27 citation statements)

References 44 publications

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“…Finally, we will have to assess in vivo if over-expression of transgenes in erythroid precursor cells can have an effect on the HSCs niche in the bone marrow or on erythrocyte differentiation, half-life and clearance 57 . Previous experiments using LV to express different proteins from erythrocytes did not show any impact on erythropoiesis [15][16][17]58 ; however, transgene-specific effects should be carefully evaluated.…”

Section: Discussionmentioning

confidence: 97%

Ex vivo editing of human hematopoietic stem cells for erythroid expression of therapeutic proteins

et al. 2020

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Targeted genome editing has a great therapeutic potential to treat disorders that require protein replacement therapy. To develop a platform independent of specific patient mutations, therapeutic transgenes can be inserted in a safe and highly transcribed locus to maximize protein expression. Here, we describe an ex vivo editing approach to achieve efficient gene targeting in human hematopoietic stem/progenitor cells (HSPCs) and robust expression of clinically relevant proteins by the erythroid lineage. Using CRISPR-Cas9, we integrate different transgenes under the transcriptional control of the endogenous α-globin promoter, recapitulating its high and erythroid-specific expression. Erythroblasts derived from targeted HSPCs secrete different therapeutic proteins, which retain enzymatic activity and cross-correct patients' cells. Moreover, modified HSPCs maintain long-term repopulation and multilineage differentiation potential in transplanted mice. Overall, we establish a safe and versatile CRISPR-Cas9-based HSPC platform for different therapeutic applications, including hemophilia and inherited metabolic disorders.

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Section: Discussionmentioning

confidence: 97%

Ex vivo editing of human hematopoietic stem cells for erythroid expression of therapeutic proteins

et al. 2020

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“…It could be provided as an outpatient treatment. Its efficacy has been demonstrated in several murine disease models, including b-thalassemia and hemophilia A [151,179], and its safety is currently tested in NHPs. With more gene therapy products on the horizon (such as hemophilia), the application of in vivo HSC transduction could extrapolate genetic treatments to a larger patient population.…”

Section: Discussionmentioning

confidence: 99%

“…SB1009 appeared to be superior to the PB system that exhibited a preference for integrations in regions surrounding transcriptional start sites and within long terminal repeat elements [149]. We have demonstrated the efficacy of SB1009mediated integration into HSCs by HDAd5/35++ vectors in several disease models, including b-hemoglobinopathies, hemophilia A, and Fanconi anemia (FA) [150,151]. More studies have reported gene transfer by Ad vectors expressing other transposon systems, such as PB [152], T2TP [153], and bacteriophagederived integrase PhiC31 [154].…”

Section: Random Integration Mediated By Transposasementioning

confidence: 99%

“…Instead of liver-directed gene transfer, HSC-directed F8 gene transfer has been investigated and showed advantages in tackling pre-existing immunity [237]. We recently reported on an approach for HSCderived, erythroid-specific F8 production based on HDAd5/35++ vectors [151]. It has been shown that the abundance and systemic distribution of erythroid cells can be harnessed for high-level production of therapeutic proteins with induced immune tolerance [238].…”

Section: Hemophiliamentioning

confidence: 99%

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Adenovirus vectors in hematopoietic stem cell genome editing

Lieber

2019

FEBS Letters

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Genome editing of hematopoietic stem cells (HSCs) represents a therapeutic option for a number of hematological genetic diseases, as HSCs have the potential for self-renewal and differentiation into all blood cell lineages. This review presents advances of genome editing in HSCs utilizing adenovirus vectors as delivery vehicles. We focus on capsid-modified, helper-dependent adenovirus vectors that are devoid of all viral genes and therefore exhibit an improved safety profile. We discuss HSC genome engineering for several inherited disorders and infectious diseases including hemoglobinopathies, Fanconi anemia, hemophilia, and HIV-1 infection by ex vivo and in vivo editing in transgenic mice, nonhuman primates, as well as in human CD34 + cells. Mechanisms of therapeutic gene transfer including episomal expression of designer nucleases and base editors, transposase-mediated random integration, and targeted homology-directed repair triggered integration into selected genomic safe harbor loci are also reviewed.

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“…However, in addition to similar immunological challenges that could impact AAV-fVIII gene therapy efficacy, in vivo delivery of LV vectors can result in high transduction efficiency of antigen-presenting cells that can reduce transduction of target hepatocytes at given doses and consequently result in downstream activation of innate and adaptive anti-viral immune responses (31). As a result, in vivo delivery of LV vectors is still in the primitive phase of development, with a focus on tactics to overcome this additional immunological challenge (32)(33)(34)(35). Thus, AAV vectors are currently the leading vector in this category of gene therapy and are rapidly progressing through clinical trials (36).…”

Section: History Of Gene Therapy For Hemophilia Amentioning

confidence: 99%

The Immune Response to the fVIII Gene Therapy in Preclinical Models

et al. 2020

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Neutralizing antibodies to factor VIII (fVIII), referred to as "inhibitors," remain the most challenging complication post-fVIII replacement therapy. Preclinical development of novel fVIII products involves studies incorporating hemophilia A (HA) and wild-type animal models. Though immunogenicity is a critical aspect of preclinical pharmacology studies, gene therapy studies tend to focus on fVIII expression levels without major consideration for immunogenicity. Therefore, little clarity exists on whether preclinical testing can be predictive of clinical immunogenicity risk. Despite this, but perhaps due to the potential for transformative benefits, clinical gene therapy trials have progressed rapidly. In more than two decades, no inhibitors have been observed. However, all trials are conducted in previously treated patients without a history of inhibitors. The current review thus focuses on our understanding of preclinical immunogenicity for HA gene therapy candidates and the potential indication for inhibitor treatment, with a focus on product-and platform-specific determinants, including fVIII transgene sequence composition and tissue/vector biodistribution. Currently, the two leading clinical gene therapy vectors are adeno-associated viral (AAV) and lentiviral (LV) vectors. For HA applications, AAV vectors are liver-tropic and employ synthetic, high-expressing, liverspecific promoters. Factors including vector serotype and biodistribution, transcriptional regulatory elements, transgene sequence, dosing, liver immunoprivilege, and host immune status may contribute to tipping the scale between immunogenicity and tolerance. Many of these factors can also be important in delivery of LV-fVIII gene therapy, especially when delivered intravenously for liver-directed fVIII expression. However, ex vivo LV-fVIII targeting and transplantation of hematopoietic stem and progenitor cells (HSPC) has been demonstrated to achieve durable and curative fVIII production without inhibitor development in preclinical models. A critical variable appears to be pretransplantation conditioning regimens that suppress and/or ablate T cells. Additionally, we and others have demonstrated the potential of LV-fVIII HSPC and liver-directed AAV-fVIII gene therapy to eradicate pre-existing inhibitors in murine and canine models of HA, Patel et al. Preclinical Gene Therapy fVIII Immunology respectively. Future preclinical studies will be essential to elucidate immune mechanism(s) at play in the context of gene therapy for HA, as well as strategies for preventing adverse immune responses and promoting immune tolerance even in the setting of pre-existing inhibitors.

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High-level protein production in erythroid cells derived from in vivo transduced hematopoietic stem cells

Abstract: Key Points An in vivo HSC transduction/selection allows for high-level protein expression from erythroid cells without side effects on erythropoiesis. This approach that did not require ex vivo HSC manipulation and transplantation resulted in phenotypic correction of murine hemophilia A.

Cited by 20 publications

References 44 publications

Ex vivo editing of human hematopoietic stem cells for erythroid expression of therapeutic proteins

Ex vivo editing of human hematopoietic stem cells for erythroid expression of therapeutic proteins

Adenovirus vectors in hematopoietic stem cell genome editing

The Immune Response to the fVIII Gene Therapy in Preclinical Models

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