Dolcetta D, Perani L, Givogri M I, Galbiati F, Amadio S, Del Carro U, Finocchiaro G, Fanzani A, Marchesini S, Naldini L, Roncarolo M G, Bongarzone E
Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.
J Gene Med. 2006 Aug;8(8):962-71. doi: 10.1002/jgm.924.
Demyelination in globoid cell leukodystrophy (GLD) is due to a deficiency of galactocerebrosidase (GALC) activity. Up to now, in vivo brain viral gene transfer of GALC showed modest impact on disease development in Twitcher mice, an animal model for GLD. Lentiviral vectors, which are highly efficient to transfer the expression of therapeutic genes in neurons and glial cells, have not been evaluated for direct cerebral therapy in GLD mice.
Lentiviral vectors containing the untagged cDNA or the hemagglutinin (HA)-tagged cDNA for the full-length mouse GALC sequence were generated and validated in vitro. In vivo therapeutic efficacy of these vectors was evaluated by histology, biochemistry and electrophysiology after transduction of ependymal or subependymal layers in young Twitcher pups.
Both GALC lentiviral vectors transduced neurons, oligodendrocytes and astrocytes with efficiencies above 75% and conferred high levels of enzyme activity. GALC accumulated in lysosomes of transduced cells and was also secreted to the extracellular medium. Conditioned GALC medium was able to correct the enzyme deficiency when added to non-transduced Twitcher glial cultures. Mice that received intraventricular injections of GALC vector showed accumulation of GALC in ependymal cells but no diffusion of the enzyme from the ependymal ventricular tree into the cerebral parenchyma. Significant expression of GALC-HA was detected in neuroglioblasts when GALC-HA lentiviral vectors were injected in the subventricular zone of Twitcher mice. Life span and motor conduction in both groups of treated Twitcher mice were not significantly ameliorated.
Lentiviral vectors showed to be efficient for reconstitution of the GALC expression in Twitcher neural cells. GALC was able to accumulate in lysosomes as well as to enter the secretory pathway of lysosomal enzymes, two fundamental aspects for gene therapy of lysosomal storage diseases. Our in vivo results, while showing the capacity of lentiviral vectors to transfer expression of therapeutic GALC in the Twitcher brain, did not limit progression of disease in Twitchers and highlight the need to evaluate other routes of administration.
球状细胞脑白质营养不良（GLD）中的脱髓鞘是由于半乳糖脑苷脂酶（GALC）活性缺乏所致。到目前为止，GALC的体内脑病毒基因转移对Twitcher小鼠（一种GLD动物模型）的疾病发展影响不大。慢病毒载体在神经元和神经胶质细胞中高效转移治疗性基因的表达，但尚未在GLD小鼠中评估其对直接脑治疗的效果。
构建了包含全长小鼠GALC序列的无标签cDNA或血凝素（HA）标签cDNA的慢病毒载体，并在体外进行了验证。在年轻的Twitcher幼崽的室管膜或室管膜下层转导后，通过组织学、生物化学和电生理学评估这些载体的体内治疗效果。
两种GALC慢病毒载体转导神经元、少突胶质细胞和星形胶质细胞的效率均高于75%，并赋予高水平的酶活性。GALC在转导细胞的溶酶体中积累，也分泌到细胞外培养基中。当将条件性GALC培养基添加到未转导的Twitcher神经胶质细胞培养物中时，能够纠正酶缺乏。接受脑室内注射GALC载体的小鼠在室管膜细胞中显示GALC积累，但酶未从室管膜脑室树扩散到脑实质中。当将GALC-HA慢病毒载体注射到Twitcher小鼠的脑室下区时，在神经胶质母细胞中检测到GALC-HA的显著表达。两组治疗的Twitcher小鼠的寿命和运动传导均未得到显著改善。
慢病毒载体显示出在Twitcher神经细胞中有效重建GALC表达的能力。GALC能够在溶酶体中积累，并进入溶酶体酶的分泌途径，这是溶酶体贮积病基因治疗的两个基本方面。我们的体内结果虽然显示了慢病毒载体在Twitcher脑中转移治疗性GALC表达的能力，但并未限制Twitcher小鼠疾病的进展，并突出了评估其他给药途径的必要性。
