In utero gene transfer and expression: a sheep transplantation model

Abstract

Retroviral-mediated gene transfer was used to insert a Neo R gene into fetal sheep hematopoietic cells obtained by exchange transfusion from lambs in utero. After gene transfer the cells were returned to the donor fetus. The lambs were examined after birth for the presence of a functioning Neo R gene. Of ten analyzable animals, six were positive for G418 resistant progenitor cells (CFU-Mix, CFU-C, BFU-E, CFU-E). Two animals were studied for extended periods of time: 8 and 24 months. Each has demonstrated a pattern wherein positive periods are interspersed with times when there were no detectable G418-resistant cells. We conclude that retroviral-mediated gene transfer can be used to insert genes into early progenitor cells of fetal sheep in utero and that the animals can continue to demonstrate blood cells expressing the gene for more than 2 years after birth. This is a US government work. There are no restrictions on its use.

In utero transfer and expression of exogenous genes in sheep

“…After birth, the Neo R sequence was detected in marrow and blood of many of these animals by PCR, and many of the animals contained G418-resistant hematopoietic progenitors of erythroid (CFU-E, BFU-E), myeloid (CFU-GM), and mix (CFU-Mix) phenotype, suggesting that primitive HSC may have been transduced. That HSC were transduced was further substantiated by the continued presence of G418resistant progenitors and PCR positivity in two animals that were observed for up to 43 and 59 months after birth [25]. This and the results of the direct-vector injection approach reported previously [1] and described here demonstrate that an in utero approach to gene therapy could result in the transfer and long-term expression of the vector-encoded genes.…”

“…These trials have thus far demonstrated gene transfer and expression in significantly higher percentages of circulating leukocytes than previous trials performed on adult patients. Moreover, in vitro studies have demonstrated that more efficient gene transfer with retroviral vectors can occur in populations of hematopoietic progenitors from fetal donors when compared to adult cell populations [25,26].…”

“…Conceivably, if transduction of even small numbers of primitive HSC could be accomplished early in gestation, expansion of transduced HSC could lead to significant transgene activity. In this regard, we have previously reported the successful transfer and long-term expression of bacterial Neo R gene in sheep following an in utero HSC transplantation/retroviral transduction protocol [25,26]. The procedure involved collecting peripheral blood from 110-day-old fetal sheep (term:145 days) by exchange transfusion with maternal blood, exposing the mononuclear cells to the N2 retroviral vector overnight, and then re-infusing these cells into the fetus.…”

The Role of the Donor in the Repair of the Marrow Vascular Niche Following Hematopoietic Stem Cell Transplant

“…Simplistically, disruption of normal homeostatic barriers leads to "holes in the levy," resulting in the loss of homeostatic integrity of the bone marrow. These holes lead to hemorrhage of nonreticulated erythrocytes into the hematopoietic compartment of the marrow space [9,11,12], the escape of marrow elements to the peripheral circulation [13], and the circulation of hematopoietic stem and progenitor cells that normally reside in the bone marrow [14]. The spleen temporarily becomes a major site of hematopoiesis until homeostasis is restored in the marrow [15], and progenitors traffic from the marrow to the spleen [16].…”

The use of umbilical cord blood as a cellular source for correction of genetic diseases affecting the hematopoietic system

“…The in vivo proliferation of retroviralmarked cord blood cells has been evaluated in the ovine system [74]. Anderson and colleagues isolated ovine fetal cord blood cells via cordocentesis, transduced the cells in vitro with a retrovirus encoding neo, and then returned the cells to the fetus.…”