Jan A. Nolta

Long-term persistence of donor nuclei in a Duchenne muscular dystrophy patient receiving bone marrow transplantation

Duchenne muscular dystrophy (DMD) is a severe progressive muscle-wasting disorder caused by mutations in the dystrophin gene. Studies have shown that bone marrow cells transplanted into lethally irradiated mdx mice, the mouse model of DMD, can become part of skeletal muscle myofibers. Whether human marrow cells also have this ability is unknown. Here we report the analysis of muscle biopsies from a DMD patient (DMD-BMT1) who received bone marrow transplantation at age 1 year for X-linked severe combined immune deficiency and who was diagnosed with DMD at age 12 years. Analysis of muscle biopsies from DMD-BMT1 revealed the presence of donor nuclei within a small number of muscle myofibers (0.5-0.9%). The majority of the myofibers produce a truncated, in-frame isoform of dystrophin lacking exons 44 and 45 (not wild-type). The presence of bone marrow-derived donor nuclei in the muscle of this patient documents the ability of exogenous human bone marrow cells to fuse into skeletal muscle and persist up to 13 years after transplantation.

The AFT024 stromal cell line supports long-term ex vivo maintenance of engrafting multipotent human hematopoietic progenitors

The immortalized murine stromal cell line AFT024 has been reported to maintain human hematopoietic progenitors in an undifferentiated state in vitro. In the current studies the beige/nude/xid (bnx) mouse in vivo xenograft model was used to examine the engraftment and multilineage generative potential of human hematopoietic progenitors after 2–3 weeks growth on AFT024 stroma, in comparison to primary stromal monolayers derived from post-natal human bone marrow. Eight to 12 months after transplantation of human CD34+CD38− cells from umbilical cord blood, cultured on AFT024 vs human stroma for 2–3 weeks, the murine bone marrow was harvested and analyzed for the presence of human myeloid and lymphoid cells. The mean percent engraftment of total human hematopoietic cells in the murine marrow was significantly higher after co-cultivation on AFT024 than on human stroma. Human myeloid and lymphoid lineage cells were detected in all mice. However, engraftment of myeloid lineage cells (CD33+), B lymphoid (CD19+), and T lymphoid cells (CD4+and CD8+) were significantly higher after co-cultivation of the human cells on AFT024 than on human stroma, prior to transplantation. Interestingly, the length of time in culture did not significantly affect the engraftment of the myeloid and T lymphoid lineage progenitors, but the percentage of B lymphoid lineage engraftment decreased significantly between 2 and 3 weeks of co-cultivation on both types of stroma. Cells with a primitive phenotype (CD45+/CD34−/CD38− and CD45+/CD34−/lin−) and cells with the capacity to generate secondary human CFU after recovery from the bnx bone marrow were maintained at significantly higher levels during culture on AFT024 stroma than on human stroma. The current studies demonstrate that the AFT024 murine stromal cell line supports the ex vivo survival and maintenance of human hematopoietic progenitors that are capable of long-term multilineage reconstitution for 2–3 weeks ex vivo, to levels superior to those that can be obtained using human stromal cells.

Long‐Term Cytokine Production from Engineered Primary Human Stromal Cells Influences Human Hematopoiesis in an In Vivo Xenograft Model

Human hematopoiesis can be supported in beige/nude/ XID (bnx) mice by coinjection of human bone marrow stromal cells engineered to secrete human interleukin 3 (HuIL‐3). The major limitation is a total absence of human B cell development in the mice, which could be due to supraphysiological levels of HuIL‐3 in the circulation. In an effort to obtain human B lymphoid, as well as T lymphoid and myeloid cell development in the mice, CD34+ cells were coinjected with human marrow stromal cells engineered to secrete human IL‐2, IL‐7, stem cell factor or FLT3 ligand, ± IL‐3. No single factor other than IL‐3 supported sustained human hematopoiesis in the mice, although cytokines were expressed for four to six months post‐transplantation. Production of both HuIL‐3 and IL‐7 in the mice supported extrathymic development of human T lymphocytes, but no B cells, myeloid cells, or clonogenic progenitors were detected. Human B cells were not produced from CD34+ cells in the bnx mice under any condition tested. Another limitation to the bnx/Hu system is a lack of maturation of human red blood cells, although BFU‐E are maintained. Stromal cells secreting human erythropoietin and IL‐3 were cotransplanted into mice with HuCD34+ cells and an increase in hematocrit from 40%‐45% to 80%‐85% resulted, with production of human and murine red blood cells. Unfortunately, all mice (n = 9) suffered strokes, displayed paralysis and died within three weeks. The bnx/Hu cotransplantation model provides an interesting system in which to study human hematopoietic cell differentiation under the influence of various cytokines.

Retroviral-mediated transfer of the human glucocerebrosidase gene into cultured Gaucher bone marrow.

Gaucher disease, a lysosomal glycolipid storage disorder, results from the genetic deficiency of an acidic glucosidase, glucocerebrosidase (GC). The beneficial effects of allogeneic bone marrow transplantation (BMT) for Gaucher disease suggest that GC gene transduction and the transplantation of autologous hematopoietic stem cells (gene therapy) may similarly alleviate symptoms. We have constructed a retroviral vector, L-GC, produced by a clone of the amphotropic packaging cell line PA317, which transduces the normal human GC cDNA with high efficiency. Whole-marrow mononuclear cells and CD34-enriched cells from a 4-yr-old female with type 3 Gaucher disease were transduced by the L-GC vector and studied in long-term bone marrow culture (LTBMC). Prestimulation of marrow with IL-3 and IL-6, followed by co-cultivation with vector-producing fibroblasts, produced gene transfer into 40-45% of the hematopoietic progenitor cells. The levels of GC expression in progeny cells (primarily mature myelomonocytic) produced by the LTBMC were quantitatively analyzed by Northern blot, Western blot, and glucocerebrosidase enzyme assay. Normal levels of GC RNA, immunoreactive protein, and enzymatic activity were detected throughout the duration of culture. These studies demonstrate that retroviral vectors can efficiently transfer the GC gene into long-lived hematopoietic progenitor cells from the bone marrow of patients with Gaucher disease and express physiologically relevant levels of GC enzyme activity.

The number and generative capacity of human B lymphocyte progenitors, measured in vitro and in vivo , is higher in umbilical cord blood than in adult or pediatric bone marrow

The lack of human B lymphocyte development in beige/nude/XID (bnx) mice is in sharp contrast to the robust development observed in another immune deficient strain, the NOD/SCID mouse. The ability to generate human B lymphocytes in the NOD/SCID, but not bnx mouse has been hypothesized to be caused by differences in the microenvironments or systemic cytokine concentrations. In the current studies we report that the differences in development can be primarily attributed to the source of the progenitors transplanted into the mice. The prior studies in bnx mice used cultured pediatric or adult bone marrow (BM) as the source of the CD34+ cells, whereas the NOD/SCID studies have predominantly used fresh or cultured umbilical cord blood (UCB). We have analyzed BM and UCB for the number of human CD34+/CD38− cells capable of in vitro B lymphocyte development, and have found a lower frequency of B lymphocyte generation in BM. The individual B lymphocyte clones that developed from bone marrow produced 100-fold fewer cells than the UCB-derived clones. In agreement with the in vitro studies, human B lymphocytes developed in bnx mice from both CD34+ and CD34+/CD38− cells isolated from human umbilical cord blood, but not from equivalent numbers of CD34+ and CD34+/CD38− progenitors from bone marrow. Therefore, the lower generative capacity, and frequency of B lymphocyte precursors in human marrow may be responsible for the previous results that showed a lack of B lymphocyte development in bnx mice.

Immunodeficient mice as models of human hematopoietic stem cell engraftment

The past year has brought forth some exciting developments in the use of murine xenotransplantation systems to study the biology and transduction of human hematopoietic stem cells. The effects of cytokines have been studied by injection into the mice or by treatment of the cell inoculum prior to injection. The importance of the cell cycle and integrin expression has been evaluated. New methods of gene therapy have been tested in xenograft models - including cell cycle manipulation and a promising new lentiviral vector system, based on HIV.

Leaky ribosomal scanning in mammalian genomes: significance of histone H4 alternative translation in vivo

Like alternative splicing, leaky ribosomal scanning (LRS), which occurs at suboptimal translational initiation codons, increases the physiological flexibility of the genome by allowing alternative translation. Comprehensive analysis of 22u2009208 human mRNAs indicates that, although the most important positions relative to the first nucleotide of the initiation codon, −3 and +4, are usually such that support initiation (A−3 = 42%, G−3 = 36% and G+4 = 47%), only 37.4% of the genes adhere to the purine (R)−3/G+4 rule at both positions simultaneously, suggesting that LRS may occur in some of the remaining (62.6%) genes. Moreover, 12.5% of the genes lack both R−3 and G+4, potentially leading to sLRS. Compared with 11 genes known to undergo LRS, 10 genes with experimental evidence for high fidelity A+1T+2G+3 initiation codons adhered much more strongly to the R−3/G+4 rule. Among the intron-less histone genes, only the H3 genes adhere to the R−3/G+4 rule, while the H1, H2A, H2B and H4 genes usually lack either R−3 or G+4. To address in vivo the significance of the previously described LRS of H4 mRNAs, which results in alternative translation of the osteogenic growth peptide, transgenic mice were engineered that ubiquitously and constitutively express a mutant H4 mRNA with an A+1→T+1 mutation. These transgenic mice, in particular the females, have a high bone mass phenotype, attributable to increased bone formation. These data suggest that many genes may fulfill cryptic functions by LRS.

15 – Haematopoietic stem cells for gene therapy

This chapter discusses the use of haematopoietic stem cells for gene therapy. Treatment of genetic disease in the haematopoietic system by gene therapy is theoretically possible if the error in blood cell formation or function results from defective activity of a single gene. If a normal counterpart of the defective gene has been cloned, it may be introduced into hematopoietic stem cells from the affected patient, then returned in an autologous bone marrow transplant. Gene therapy via hematopoietic stem cells may ultimately be used to treat sickle cell anemia, thalassemia, congenital immune deficiencies, lysosomal storage disorders, and Fanconis anemia. Possible future targets for gene therapy are leukemia and acquired immune deficiency syndrome, although the treatment will be more complex than a simple gene replacement. This chapter discusses the murine models for gene therapy. It explains concepts related to large animal models of gene therapy. It also describes human cells in vitro , stromal support, and xenograft systems. An overview of clinical studies using gene marking is also presented in this chapter.

Extrathymic human t lymphocyte development and regulation in immune deficient mice

Abstract We previously studied extrathymic human T lymphocyte development from stem/progenitor subsets in the bone marrow of bnx mice. Current studies examined human T cell development from purified CD34 + CD3 − stem cells in a new strain that we developed, nude/NOD/SCID mice. Human T cells isolated from bnx mice contained double CD4 + /CD8 + and single CD3 + /CD4 + or CD3 + /CD8 + cells, but they were anergic to activation by PMA plus ionomycin (P+1) and to CD3 and CD28 crosslinking. In nude/NOD/SCID mice, there was more variability in human T cell development in the marrow. Double positive CD4/CD8 and CD4 + /cytoCD3 + populations, which expressed CD3 mRNA, were found in some of the mice. But no CD8 single positive cells were found in any mice. In some mice, only myeloid progenitor cells and B cells were found, much like the NOD/SCID strain. The differences may be due to different microenvironments or to the types of human cells engrafted in the starting populations. Studying the influence of implanting human pediatric thymus slices into nude/NOD/SCID on the development of human T cells is in progress. Thymus grafts get well vascularized in the peritoneal cavity of mice. Early studies suggest that the presence of the thymus makes the human T cells less hyporesponsive. Future studies will detemine whether the human cells are being selected in the thymus or whether thymic stromal factors are influencing development and/or activation of human T cells.

Gene Therapy with Cord Blood Hematopoietic Stem Cells for Adenosine Deaminase Deficiency: An Update

Gene therapy for ADA deficiency has been a model disease in which to evaluate hematopoietic stem cells for clinical gene therapy [1]. ADA deficiency has been a candidate disease because n n1. n nADA is a single polypeptide protein, n n n n n2. n na clinically normal phenotype is associated with a wide range of enzymatic function (5–500% of normal), and n n n n n3. n nADA is widely expressed so that tissue specific expression is not required.