Ypt/Rab proteins constitute the largest subfamily of the Ras superfamily of monomeric GTPases and are regulators of vesicular protein transport. Their slow intrinsic GTPase activity (10(-4)-10(-3) min(-1) at 30 degrees C) has to be accelerated to switch the active to the inactive conformation. We have identified the catalytic domain within the C-terminal halves of two yeast GTPase-activating proteins (GAPs), Gyp1p and Gyp7p, with specificity for Ypt/Rab GTPases. The catalytically active fragments of Gyp1p and Gyp7p were more active than the full-length proteins and accelerated the intrinsic GTP hydrolysis rates of their preferred substrates by factors of 4.5 x 10(4) and 7.8 x 10(5), respectively. The K(m) values for the Gyp1p and Gyp7p active fragments (143 and 42 microM, respectively) indicate that the affinities of those GAPs for their substrates are very low. The catalytic domains of Gyp1p and Gyp7p contain five invariant arginine residues; substitutions of only one of them (R343 in Gyp1p and R458 in the analogous position of Gyp7p) rendered the GAPs almost completely inactive. We suggest that Ypt/Rab-GAPs, like Ras- and Rho-GAPs, follow the same mode of action and provide a catalytic arginine ('arginine finger') in trans to accelerate the GTP hydrolysis rate of the transport GTPases.

Severe adverse events related to insertional mutagenesis have reinforced interest in self-inactivating (SIN) retroviral vectors lacking enhancer-promoter sequences in the long terminal repeats (LTRs). Here, we have compared the potency of gammaretroviral and lentiviral vectors expressing the P140K mutant of O(6)-methylguanine-DNA methyltransferase (MGMT). MGMT-P140K is a clinically relevant selection marker that mediates a strong survival advantage in hematopoietic cells exposed to alkylating agents. We designed gammaretroviral and lentiviral vectors that contained identical enhancer-promoter sequences located either in the LTR or downstream of the packaging region, for internal initiation of transcription from SIN backbones. Gammaretroviral vectors with intact LTRs containing enhancer-promoter sequences showed both higher titers and higher expression levels than the lentiviral counterparts, likely a result of suboptimal RNA processing of the lentiviral leader region. In the SIN context, gammaretroviral and lentiviral vectors with comparable internal cassettes had similar expression properties. Interestingly, gammaretroviral SIN vectors pseudotyped with RD114/TR had a higher transduction efficiency on proliferating human CD34(+) cells than lentiviral counterparts. These results encourage further investigations into the formation of retroviral hybrid vectors that combine the desired properties of high efficiency and increased biosafety.

Ectopic retroviral expression of homeobox B4 (HOXB4) causes an accelerated and enhanced regeneration of murine hematopoietic stem cells (HSCs) and is not known to compromise any program of lineage differentiation. However, HOXB4 expression levels for expansion of human stem cells have still to be established. To test the proposed hypothesis that HOXB4 could become a prime tool for in vivo expansion of genetically modified human HSCs, we retrovirally overexpressed HOXB4 in purified cord blood (CB) CD34 ؉ cells together with green fluorescent protein (GFP) as a reporter protein, and evaluated the impact of ectopic HOXB4 expression on proliferation and differentiation in vitro and in vivo. When injected separately into nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice or in competition with control vector-transduced cells, HOXB4-overexpressing cord blood CD34 ؉ cells had a selective growth advantage in vivo, which resulted in a marked enhancement of the primitive CD34 ؉ subpopulation (P ‫؍‬ .01). However, high HOXB4 expression substantially impaired the myeloerythroid differentiation program, and this was reflected in a severe reduction of erythroid and myeloid progenitors in vitro (P < .03) and in vivo (P ‫؍‬ .01). Furthermore, HOXB4 overexpression also significantly reduced B-cell output (P < .01). These results show for the first time unwanted side effects of ectopic HOXB4 expression and therefore underscore the need to carefully determine the therapeutic window of HOXB4 expression levels before initializing clinical