Christina Ironside

Long-term polyclonal and multilineage engraftment of methylguanine methyltransferase P140K gene-modified dog hematopoietic cells in primary and secondary recipients

Overexpression of methylguanine methyltransferase P140K (MGMTP140K) has been successfully used for in vivo selection and chemoprotection in mouse and large animal studies, and has promise for autologous and allogeneic gene therapy. We examined the long-term safety of MGMTP140K selection in a clinically relevant dog model. Based on the association of provirus integration and proto-oncogene activation leading to leukemia in the X-linked immunodeficiency trial, we focused our analysis on the distribution of retrovirus integration sites (RIS) relative to proto-oncogene transcription start sites (TSS). We analyzed RIS near proto-oncogene TSS before (n = 157) and after (n = 129) chemotherapy in dogs that received MGMTP140K gene-modified cells and identified no overall increase of RIS near proto-oncogene TSS after chemotherapy. We also wanted to determine whether in vivo selected cells retained fundamental characteristics of hematopoietic stem cells. To that end, we performed secondary transplantation of MGMTP140K gene-modified cells after in vivo selection in dog leukocyte antigen (DLA)-matched dogs. Gene-modified cells achieved multilineage repopulation, and we identified the same gene-modified clone in both dogs more than 800 and 900 days after transplantation. These data suggest that MGMTP140K selection is well tolerated and should allow clinically for selection of gene-corrected cells in genetic or infectious diseases or chemoprotection for treatment of malignancy.

Cocal-pseudotyped Lentiviral Vectors Resist Inactivation by Human Serum and Efficiently Transduce Primate Hematopoietic Repopulating Cells

Lentiviral vectors are established as efficient and convenient vehicles for gene transfer. They are almost always pseudotyped with the envelope glycoprotein of vesicular stomatitis virus (VSV-G) due to the high titers that can be achieved, their stability, and broad tropism. We generated a novel cocal vesiculovirus envelope glycoprotein plasmid and compared the properties of lentiviral vectors pseudotyped with cocal, VSV-G, and a modified feline endogenous retrovirus envelope glycoprotein (RD114/TR). Cocal-pseudotyped lentiviral vectors can be produced at titers as high as with VSV-G, have a broad tropism, and are stable, allowing for efficient concentration by centrifugation. Additionally, cocal vectors are more resistant to inactivation by human serum than VSV-G-pseudotyped vectors, and efficiently transduce human CD34(+) nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse-repopulating cells (SRCs), and long-term primate hematopoietic repopulating cells. These studies establish the potential of cocal-pseudotyped lentiviral vectors for a variety of scientific and therapeutic gene transfer applications, including in vivo gene delivery and hematopoietic stem cell (HSC) gene therapy.

Stem Cell Selection In Vivo Using Foamy Vectors Cures Canine Pyruvate Kinase Deficiency

Background Hematopoietic stem cell (HSC) gene therapy has cured immunodeficiencies including X-linked severe combined immunodeficiency (SCID-X1) and adenine deaminase deficiency (ADA). For these immunodeficiencies corrected cells have a selective advantage in vivo, and low numbers of gene-modified cells are sufficient to provide therapeutic benefit. Strategies to efficiently transduce and/or expand long-term repopulating cells in vivo are needed for treatment of diseases that require higher levels of corrected cells, such as hemoglobinopathies. Here we expanded corrected stem cells in vivo in a canine model of a severe erythroid disease, pyruvate kinase deficiency. Methodology/Principal Findings We used a foamy virus (FV) vector expressing the P140K mutant of methylguanine methyltransferase (MGMTP140K) for in vivo expansion of corrected hematopoietic repopulating cells. FV vectors are attractive gene transfer vectors for hematopoietic stem cell gene therapy since they efficiently transduce repopulating cells and may be safer than more commonly used gammaretroviral vectors. Following transplantation with HSCs transduced ex vivo using a tri-cistronic FV vector that expressed EGFP, R-type pyruvate kinase, and MGMTP140K, we were able to increase marking from approximately 3.5% to 33% in myeloid long-term repopulating cells resulting in a functional cure. Conclusions/Significance Here we describe in one affected dog a functional cure for a severe erythroid disease using stem cell selection in vivo. In addition to providing a potential cure for patients with pyruvate kinase deficiency, in vivo selection using foamy vectors with MGMTP140K has broad potential for several hematopoietic diseases including hemoglobinopathies.

In vivo selection of autologous MGMT gene-modified cells following reduced-intensity conditioning with BCNU and temozolomide in the dog model

Chemotherapy with 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) and temozolomide (TMZ) is commonly used for the treatment of glioblastoma multiforme (GBM) and other cancers. In preparation for a clinical gene therapy study in patients with glioblastoma, we wished to study whether these reagents could be used as a reduced-intensity conditioning regimen for autologous transplantation of gene-modified cells. We used an MGMT(P140K)-expressing lentivirus vector to modify dog CD34+ cells and tested in four dogs whether these autologous cells engraft and provide chemoprotection after transplantation. Treatment with O6-benzylguanine (O6BG)/TMZ after transplantation resulted in gene marking levels up to 75%, without significant hematopoietic cytopenia, which is consistent with hematopoietic chemoprotection. Retrovirus integration analysis showed that multiple clones contribute to hematopoiesis. These studies demonstrate the ability to achieve stable engraftment of MGMT(P140K)-modified autologous hematopoietic stem cells (HSCs) after a novel reduced-intensity conditioning protocol using a combination of BCNU and TMZ. Furthermore, we show that MGMT(P140K)-HSC engraftment provides chemoprotection during TMZ dose escalation. Clinically, chemoconditioning with BCNU and TMZ should facilitate engraftment of MGMT(P140K)-modified cells while providing antitumor activity for patients with poor prognosis glioblastoma or alkylating agent-sensitive tumors, thereby supporting dose-intensified chemotherapy regimens.

Stable marking and transgene expression without progression to monoclonality in canine long-term hematopoietic repopulating cells transduced with lentiviral vectors.

Lentiviral gene transfer vectors have a number of potential advantages over gammaretroviral vectors including more efficient transduction of nondividing cells, a more favorable integration site profile, and the ability to accommodate large transgenes. Here, we present long-term follow-up data of animals that received lentivirus-transduced CD34-enriched cells. Six long-term surviving dogs were available for analysis. Transgene expression was analyzed from at least 12 months to more than 5 years after transplantation in peripheral blood cells and multiple cell lineages. All animals demonstrated long-term stable transgene expression in peripheral blood myeloid, lymphoid, and red blood cells as well as in platelets. Vector integration sites were analyzed by linear amplification-mediated polymerase chain reaction and showed a polyclonal repopulation pattern in all animals. There was no evidence of any development of monoclonality or leukemia in the animals. The stable long-term multilineage transgene expression, together with detection of the same integration site in myeloid and lymphoid cells, strongly suggests the transduction of long-term repopulating stem cells. Our data demonstrate safe and efficient transduction of multilineage long-term repopulating cells with lentiviral vectors and support the use of such vectors for gene therapy studies in patients.

A retroviral vector common integration site between leupaxin and zinc finger protein 91 (ZFP91) observed in baboon hematopoietic repopulating cells

OBJECTIVE Retroviral vector proviruses can lead to aberrant expression of nearby genes in hematopoietic repopulating cells, leading to an over-representation of clones with dysregulated genes that affect hematopoiesis. Common integration sites (CISs) identified using the vector provirus as a molecular tag can be used to identify these genes. Here we characterized a retroviral CIS observed at high frequency in baboon hematopoietic repopulating cells that has not been described previously. MATERIALS AND METHODS Gammaretroviral vector integration sites in baboon repopulating cells identified by polymerase chain reaction amplification were localized to the human genome to identify a CIS. The presence of each clone was tracked over time using allele-specific polymerase chain reaction. RESULTS In three different animals that received gammaretrovirally transduced CD34-enriched bone marrow cells, vector proviruses were identified at three distinct sites within a window of 664 base pairs between leupaxin and zinc finger protein 91 (ZFP91). All three integrants of the CIS occurred within a CpG island between leupaxin and zinc finger protein 91 (ZFP91). CONCLUSIONS We describe a novel CIS between leupaxin and ZFP91 in hematopoietic repopulating cells. Our data suggest that leupaxin and/or ZFP91 may play a role in hematopoietic repopulating cells.

In Vivo Murine-Matured Human CD3+ Cells as a Preclinical Model for T Cell-Based Immunotherapies

Adoptive cellular immunotherapy is a promising and powerful method for the treatment of a broad range of malignant and infectious diseases. Although the concept of cellular immunotherapy was originally proposed in the 1990s, it has not seen successful clinical application until recent years. Despite significant progress in creating engineered receptors against both malignant and viral epitopes, no efficient preclinical animal models exist for rapidly testing and directly comparing these engineered receptors. The use of matured human T cells in mice usually leads to graft-versus-host disease (GvHD), which severely limits the effectiveness of such studies. Alternatively, adult apheresis CD34+ cells engraft in neonatal non-obese diabetic (NOD)-severe combined immunodeficiency (SCID)-common γ chain–/– (NSG) mice and lead to the development of CD3+ T cells in peripheral circulation. We demonstrate that these in vivo murine-matured autologous CD3+ T cells from humans (MATCH) can be collected from the mice, engineered with lentiviral vectors, reinfused into the mice, and detected in multiple lymphoid compartments at stable levels over 50 days after injection. Unlike autologous CD3+ cells collected from human donors, these MATCH mice did not exhibit GvHD after T cell administration. This novel mouse model offers the opportunity to screen different immunotherapy-based treatments in a preclinical setting.

In vivo protection of activated Tyr22-dihydrofolate reductase gene-modified canine T lymphocytes from methotrexate.

Nonmyeloablative allogeneic hematopoietic stem cell (HSC) transplantation can cure malignant and nonmalignant diseases affecting the hematopoietic system, such as severe combined immunodeficiencies, aplastic anemia and hemoglobinopathies. Although nonmyeloablative is favored over myeloablative transplantation for many patients, graft rejection remains problematic. One strategy for decreasing rejection is to protect donor activated T cells in the graft from methotrexate (MTX) by genetically modifying the cells to express MTX‐resistant dihydrofolate reductase (Tyr22‐DHFR), leaving the immunosuppressive effects of MTX to act solely on activated host T lymphocytes, shifting the balance to favor allogeneic engraftment.

HIV infection results in clonal expansions containing integrations within pathogenesis-related biological pathways

The genomic integration of HIV into cells results in long-term persistence of virally infected cell populations. This integration event acts as a heritable mark that can be tracked to monitor infected cells that persist over time. Previous reports have documented clonal expansion in people and have linked them to proto-oncogenes; however, their significance or contribution to the latent reservoir has remained unclear. Here, we demonstrate that a directed pattern of clonal expansion occurs in vivo, specifically in gene pathways important for viral replication and persistence. These biological processes include cellular division, transcriptional regulation, RNA processing, and posttranslational modification pathways. This indicates preferential expansion when integration events occur within genes or biological pathways beneficial for HIV replication and persistence. Additionally, these expansions occur quickly during unsuppressed viral replication in vivo, reinforcing the importance of early intervention for individuals to limit reservoir seeding of clonally expanded HIV-infected cells.

Engineering resistance to CD33-targeted immunotherapy in normal hematopoiesis by CRISPR/Cas9-deletion of CD33 exon 2

CD33 has long been pursued as immunotherapeutic target in acute myeloid leukemia (AML) [1, 2]. Improved survival with gemtuzumab ozogamicin (GO) validates this approach [3]. Partly stimulated by GO’s success, several investigational CD33-directed therapeutics are currently in clinical testing [4]. However, CD33 expression on normal hematopoietic cells leads to “on-target, off-leukemia” toxicity with significant morbidity/mortality from profound cytopenias, limiting the use of CD33-directed immunotherapies [4]. This toxicity should be minimal if normal blood cells did not express the epitope targeted by these antibodies. Supporting the feasibility of CD33-engineering the hematopoietic system are the findings that CD33-deficient mice have a very mild phenotype and show no difference in cellular response to pro-inflammatory stimuli compared to wild-type animals, indicating functional degeneracy between CD33 and other proteins [5]. Moreover, recent studies have shown that CRISPR/Cas9-mediated disruption of the CD33 coding region in CD34+ hematopoietic stem and progenitor cells (HSPCs) may not affect engraftment [6], suggesting that the generation of CD33-manipulated hematopoiesis is a clinically viable strategy to protect from “off-leukemia” cell toxicity of CD33-directed immunotherapy. Here we have investigated an alternative, precise CD33 genome-editing approach that would only eliminate exon 2 and therefore the V-set immunoglobulin-like domain, which is the target of all current clinical CD33directed approaches. Our editing strategy is expected to result in expression of a naturally occurring shorter isoform of CD33 (CD33) but not full-length CD33 (CD33), which may minimize potential adverse effects associated with disruption of the entire CD33 locus. We used CRISPR/ Cas9 [7–10] to accomplish this goal and functionally assessed genome-edited human hematopoietic cells in vitro and in immunodeficient mice. Human myeloid ML-1 cells and human fetal liver CD34 + HSPCs were used for our studies. ML-1 cells were maintained as described [11]. Human fetal liver CD34+ cells were enriched by immunomagnetic separation from tissue obtained from Advance Bioscience Resources Inc. (ABR, Alameda, CA). Cells were cultured in StemSpan SFEMII media (StemCell Technologies, Cambridge, WA) supplemented with penicillin/streptomycin (Life Technologies, Carlsbad, CA), Stem cell factor , Thrombopoietin (both PeproTech, Rocky Hill, NJ), and FLT3-L (Miltenyi Biotec, Auburn, CA). CRISPR/Cas9-editing was carried out by electroporation of purified Cas9 protein (TrueCut Cas9 V2; ThermoFisher Scientific, Waltham, MA) complexed with synthetic guide RNAs (sgRNAs; Supplementary Table 1), which were modified at the 5′ and 3′ ends with 2′O-methyl-3′-phosphorothiate (Synthego, Redwood City, CA) using the ECM 380 Square Wave Electroporation system (Harvard Apparatus, Cambridge, MA) [12]. For evaluation of colony-forming units (CFUs), 1500 CD34+ cells were seeded in 3.5 mL ColonyGEL 1402 (ReachBio, Seattle, WA) and scored after 12–14 days. CFU DNA was extracted in QuickExtract (Epicentre, Madison, WI). We quantified drug-induced cytotoxicity as described previously [11, 13]. Briefly, parental and CRISPRengineered ML-1 cells were incubated in 96-well round These authors contributed equally: Olivier Humbert, George S. Laszlo and Hans-Peter Kiem, Roland B. Walter