Differential effects of HOXB4 and NUP98-HOXA10hd on hematopoietic repopulating cells in a nonhuman primate model.

Expansion of hematopoietic stem cells (HSCs) is beneficial in settings where HSC numbers are limited, such as cord blood transplantation. The human homeobox transcription factor HOXB4 has been shown to enhance stem cell expansion in several experimental models. We have shown previously that HOXB4 overexpression in monkey CD34(+) cells has a dramatic effect on expansion and engraftment of short-term repopulating cells. Here, we wished to compare the effects of HOXB4 and another candidate gene, NUP98-HOXA10hd (NA10hd). We used a competitive repopulation assay in pigtailed macaques to study engraftment of CD34(+) cells modified with gammaretroviral HOXB4YFP or NA10hdGFP. We found that HOXB4YFP contributed more to early hematopoiesis (<30 days), whereas NA10hdGFP contributed more to later hematopoiesis. In each case, we observed two distinct peaks in engraftment of NA10hd-transduced cells, one within 20 days post transplant and another after 5-6 months. Analysis of CD14(+), CD3(+), and CD20(+) subsets confirmed that higher percentages of cells of each lineage were derived from NA10hdGFP(+) progenitors than from HOXB4YFP(+) progenitors. In conclusion, we show that HOXB4 and NA10hd both have a significant impact on hematopoietic reconstitution; however, these effects are differential and therefore may offer complementary strategies for HSC expansion.

Protection of stem cell-derived lymphocytes in a primate AIDS gene therapy model after in vivo selection.

BACKGROUND: There is currently no effective AIDS vaccine, emphasizing the importance of developing alternative therapies. Recently, a patient was successfully transplanted with allogeneic, naturally resistant CCR5-negative (CCR5Delta32) cells, setting the stage for transplantation of naturally resistant, or genetically modified stem cells as a viable therapy for AIDS. Hematopoietic stem cell (HSC) gene therapy using vectors that express various anti-HIV transgenes has also been attempted in clinical trials, but inefficient gene transfer in these studies has severely limited the potential of this approach. Here we evaluated HSC gene transfer of an anti-HIV vector in the pigtailed macaque (Macaca nemestrina) model, which closely models human transplantation. METHODS AND FINDINGS: We used lentiviral vectors that inhibited both HIV-1 and simian immunodeficiency virus (SIV)/HIV-1 (SHIV) chimera virus infection, and also expressed a P140K mutant methylguanine methyltransferase (MGMT) transgene to select gene-modified cells by adding chemotherapy drugs. Following transplantation and MGMT-mediated selection we demonstrated transgene expression in over 7% of stem-cell derived lymphocytes. The high marking levels allowed us to demonstrate protection from SHIV in lymphocytes derived from gene-modified macaque long-term repopulating cells that expressed an HIV-1 fusion inhibitor. We observed a statistically significant 4-fold increase of gene-modified cells after challenge of lymphocytes from one macaque that received stem cells transduced with an anti-HIV vector (p<0.02, Student's t-test), but not in lymphocytes from a macaque that received a control vector. We also established a competitive repopulation assay in a second macaque for preclinical testing of promising anti-HIV vectors. The vectors we used were HIV-based and thus efficiently transduce human cells, and the transgenes we used target HIV-1 genes that are also in SHIV, so our findings can be rapidly translated to the clinic. CONCLUSIONS: Here we demonstrate the ability to select protected HSC-derived lymphocytes in vivo in a clinically relevant nonhuman primate model of HIV/SHIV infection. This approach can now be evaluated in human clinical trials in AIDS lymphoma patients. In this patient setting, chemotherapy would not only kill malignant cells, but would also increase the number of MGMTP140K-expressing HIV-resistant cells. This approach should allow for high levels of HIV-protected cells in AIDS patients to evaluate AIDS gene therapy.

Assessment and improvement of the Plasmodium yoelii yoelii genome annotation through comparative analysis.

MOTIVATION: The sequencing of the Plasmodium yoelii genome, a model rodent malaria parasite, has greatly facilitated research for the development of new drug and vaccine candidates against malaria. Unfortunately, only preliminary gene models were annotated on the partially sequenced genome, mostly by in silico gene prediction, and there has been no major improvement of the annotation since 2002. RESULTS: Here we report on a systematic assessment of the accuracy of the genome annotation based on a detailed analysis of a comprehensive set of cDNA sequences and proteomics data. We found that the coverage of the current annotation tends to be biased toward genes expressed in the blood stages of the parasite life cycle. Based on our proteomic analysis, we estimate that about 15% of the liver stage proteome data we have generated is absent from the current annotation. Through comparative analysis we identified and manually curated a further 510 P. yoelii genes which have clear orthologs in the P. falciparum genome, but were not present or incorrectly annotated in the current annotation. This study suggests that improvements of the current P. yoelii genome annotation should focus on genes expressed in stages other than blood stages. Comparative analysis will be critically helpful for this re-annotation. The addition of newly annotated genes will facilitate the use of P. yoelii as a model system for studying human malaria. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.