Search

Mucopolysaccharidosis III (MPSIII, Sanfilippo syndrome) is a devastating lysosomal storage disease that primarily affects the central nervous system. MPSIIIA is caused by loss-of-function mutations in the gene coding for sulfamidase (N-sulfoglucosamine sulfohydrolase/SGSH) resulting in SGSH enzyme deficiency, a buildup of heparin sulfate and subsequent neurodegeneration. There is currently no cure or disease modifying treatment for MPSIIIA. A mouse model for MPSIIIA was characterized in 1999 and later backcrossed onto the C57BL/6 background. In the present study, a novel immune deficient MPSIIIA mouse model (MPSIIIA-TKO) was created by backcrossing the immune competent, C57BL/6 MPSIIIA mouse to an immune deficient mouse model lacking Rag2, CD47 and Il2rg genes. The resulting mouse model has undetectable SGSH activity, exhibits histological changes consistent with MPSIIIA and lacks T cells, B cells and NK cells. This new mouse model has the potential to be extremely useful in testing human cellular therapies in an animal model as it retains the MPSIIIA disease phenotype while tolerating xenotransplantation., (© 2023. The Author(s).)

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.

Gaucher disease is a lysosomal storage disorder resulting form deficiency of the acid beta-glucosidase, glucocerebrosidase (GC). Allogeneic bone marrow transplantation has been beneficial in the treatment of Gaucher patients. Therefore, this disorder may be an ideal candidate for gene therapy by GC gene transduction of hematopoietic stem cells. We sought to increase the extent of gene transfer into CD34+ cells from the marrow of a Gaucher patient using G1GC, a simple retroviral vector containing a normal human GC cDNA. The ability of autologous stromal support and recombinant cytokines to increase the extent of transduction of colony-forming-cells (CFCs) and long-term culture initiating cells (LTCICs) was assessed. The presence of a stromal layer significantly increased the extent of GC gene transfer into 14-day CFCs, as determined by polymerase chain reaction (PCR) of individual colonies (18.8% with stroma versus 5% without, P < 0.001). Stromal support also increased the extent of transduction of LTCICs (10% with stroma versus 0.83% without, P < 0.001). Non-adherent cells from long-term bone marrow cultures initiated with CD34+ progenitors transduced on autologous stroma had higher levels of GC enzyme activity than cultures initiated with cells transduced without stroma. The percentage of cells which were GC positive by immunohistochemistry was also increased (21.1% with stroma versus 2.7% without, P = 0.0003). The addition of cytokines (IL-3, IL-6 and Steel factor) to the transduction, in the presence of stroma, significantly increased the extent of gene transfer into CFCs but not LTCICs. These studies indicate that the GC gene can be effectively transduced into LTCICs by retroviral vectors in the presence of stroma at levels significant for clinical gene therapy trials in patients with Gaucher disease.

The human glucocerebrosidase (GC) gene has been expressed in the progeny of murine hematopoietic stem cells following transduction of marrow with a retroviral vector (G2) containing the human GC cDNA. Murine marrow was transduced via co-cultivation following prestimulation in the presence or absence of recombinant IL-3 and IL-6. A high rate of gene transfer and expression (95%) was demonstrated in primary day 12 CFU-S foci following bone marrow transplantation (BMT) of G2-transduced marrow into lethally irradiated syngeneic recipient mice. Immunoreactive human GC protein was also documented in the CFU-S foci. Primary recipient mice were examined 4-6 months following BMT. A higher rate of gene transfer (87%) was seen in hematopoietic organs of recipients of prestimulated donor marrow compared with organs from initially unstimulated marrow (25%). A high rate of expression of human GC was also documented in the prestimulated organs (50%) when compared with the unstimulated group (25%). Secondary BMT was performed using marrow from the long-lived primary recipients. The human GC gene was present in 88% of secondary day 12 CFU-S foci examined in the prestimulated group versus 23% in the unstimulated group. Expression of the human GC gene was documented in secondary day 12 CFU-S foci, providing strong evidence of initial hematopoietic stem cell transduction.