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. 2008 Nov;95(3):133-41.
doi: 10.1016/j.ymgme.2008.08.003. Epub 2008 Sep 20.

In vivo delivery of human acid ceramidase via cord blood transplantation and direct injection of lentivirus as novel treatment approaches for Farber disease

Shobha Ramsubir  1 , Takahiro Nonaka, Carmen Bedia Girbés, Stéphane Carpentier, Thierry Levade, Jeffrey A Medin
Affiliations

In vivo delivery of human acid ceramidase via cord blood transplantation and direct injection of lentivirus as novel treatment approaches for Farber disease

Shobha Ramsubir et al. Mol Genet Metab. 2008 Nov.

Abstract

Farber disease is a rare lysosomal storage disorder (LSD) caused by a deficiency of acid ceramidase (AC) activity and subsequent accumulation of ceramide. Currently, there is no treatment for Farber disease beyond palliative care and most patients succumb to the disorder at a very young age. Previously, our group showed that gene therapy using oncoretroviral vectors (RV) could restore enzyme activity in Farber patient cells. The studies described here employ novel RV and lentiviral (LV) vectors that engineer co-expression of AC and a cell surface marking transgene product, human CD25 (huCD25). Transduction of Farber patient fibroblasts and B cells with these vectors resulted in overexpression of AC and led to a 90% and 50% reduction in the accumulation of ceramide, respectively. Vectors were also evaluated in human hematopoietic stem/progenitor cells (HSPCs) and by direct in vivo delivery in mouse models. In a xenotransplantation model using NOD/SCID mice, we found that transduced CD34(+) cells could repopulate irradiated recipient animals, as measured by CD25 expression. When virus was injected intravenously into mice, soluble CD25 was detected in the plasma and increased AC activity was present in the liver up to 14 weeks post-injection. These findings suggest that vector and transgene expression can persist long-term and offer the potential of a lasting cure. To our knowledge, this is the first report of in vivo testing of direct gene therapy strategies for Farber disease.

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Figures

FIG. 1
FIG. 1. huCD25 expression on transduced, immortalized Farber patient cells
Farber patient fibroblasts (A) and B cells (B) were transduced with either the oncoretrovirus (RV) or lentivirus (LV) engineered to express both human AC and CD25. Cells were stained with anti-huCD25-PE antibody and analyzed by flow cytometry. NT: non-transduced.
FIG. 2
FIG. 2. Lysosomal ceramide turnover and ceramide content in transduced Farber patient cells
Immortalized Farber patient cells were transduced with either oncoretrovirus (RV) or lentivirus (LV) encoding human AC and huCD25. Non-transduced and transduced Farber patient cells were pulsed with [3H-ceramide]-sphingomyelin for 48 h. Lipids were isolated, and then separated by TLC. The AC activity of fibroblasts (A) and B cells (C) are shown. To determine ceramide content, lipid extracts were incubated with E. coli diacylglycerol kinase and [γ32P]ATP. Radioactive ceramide 1-phosphate was isolated by TLC and quantified by liquid scintillation analysis for both fibroblast (B) and B cell (D) extracts. Error bars represent SD; measurements are averages of at least three separate experiments. * p < 0.05, *** p < 0.001, for groups indicated versus non-transduced (NT) controls.
FIG. 2
FIG. 2. Lysosomal ceramide turnover and ceramide content in transduced Farber patient cells
Immortalized Farber patient cells were transduced with either oncoretrovirus (RV) or lentivirus (LV) encoding human AC and huCD25. Non-transduced and transduced Farber patient cells were pulsed with [3H-ceramide]-sphingomyelin for 48 h. Lipids were isolated, and then separated by TLC. The AC activity of fibroblasts (A) and B cells (C) are shown. To determine ceramide content, lipid extracts were incubated with E. coli diacylglycerol kinase and [γ32P]ATP. Radioactive ceramide 1-phosphate was isolated by TLC and quantified by liquid scintillation analysis for both fibroblast (B) and B cell (D) extracts. Error bars represent SD; measurements are averages of at least three separate experiments. * p < 0.05, *** p < 0.001, for groups indicated versus non-transduced (NT) controls.
FIG. 2
FIG. 2. Lysosomal ceramide turnover and ceramide content in transduced Farber patient cells
Immortalized Farber patient cells were transduced with either oncoretrovirus (RV) or lentivirus (LV) encoding human AC and huCD25. Non-transduced and transduced Farber patient cells were pulsed with [3H-ceramide]-sphingomyelin for 48 h. Lipids were isolated, and then separated by TLC. The AC activity of fibroblasts (A) and B cells (C) are shown. To determine ceramide content, lipid extracts were incubated with E. coli diacylglycerol kinase and [γ32P]ATP. Radioactive ceramide 1-phosphate was isolated by TLC and quantified by liquid scintillation analysis for both fibroblast (B) and B cell (D) extracts. Error bars represent SD; measurements are averages of at least three separate experiments. * p < 0.05, *** p < 0.001, for groups indicated versus non-transduced (NT) controls.
FIG. 2
FIG. 2. Lysosomal ceramide turnover and ceramide content in transduced Farber patient cells
Immortalized Farber patient cells were transduced with either oncoretrovirus (RV) or lentivirus (LV) encoding human AC and huCD25. Non-transduced and transduced Farber patient cells were pulsed with [3H-ceramide]-sphingomyelin for 48 h. Lipids were isolated, and then separated by TLC. The AC activity of fibroblasts (A) and B cells (C) are shown. To determine ceramide content, lipid extracts were incubated with E. coli diacylglycerol kinase and [γ32P]ATP. Radioactive ceramide 1-phosphate was isolated by TLC and quantified by liquid scintillation analysis for both fibroblast (B) and B cell (D) extracts. Error bars represent SD; measurements are averages of at least three separate experiments. * p < 0.05, *** p < 0.001, for groups indicated versus non-transduced (NT) controls.
FIG. 3
FIG. 3. Metabolic cooperativity demonstrated by uptake of secreted AC by non-transduced Farber fibroblasts
(A) Non-transduced (NT) Farber fibroblasts were overlaid with media harvested from the indicated cells and incubated for 48 h. The cells were then pulsed with [3H-ceramide]-sphingomyelin for 24 h and lipids analyzed by TLC. Error bars represent SD; measurements are averages of three independent experiments. * p < 0.05, ** p < 0.01, for groups indicated versus the non-transduced (NT) control. (B) Normal, NT and transduced (RV/AC/huCD25 or LV/AC/huCD25) Farber fibroblasts were grown in serum-free culture medium. After 24 h, both cells and media were collected. Proteins were separated by PAGE and immunoblotted with an anti-acid ceramidase antibody.
FIG. 3
FIG. 3. Metabolic cooperativity demonstrated by uptake of secreted AC by non-transduced Farber fibroblasts
(A) Non-transduced (NT) Farber fibroblasts were overlaid with media harvested from the indicated cells and incubated for 48 h. The cells were then pulsed with [3H-ceramide]-sphingomyelin for 24 h and lipids analyzed by TLC. Error bars represent SD; measurements are averages of three independent experiments. * p < 0.05, ** p < 0.01, for groups indicated versus the non-transduced (NT) control. (B) Normal, NT and transduced (RV/AC/huCD25 or LV/AC/huCD25) Farber fibroblasts were grown in serum-free culture medium. After 24 h, both cells and media were collected. Proteins were separated by PAGE and immunoblotted with an anti-acid ceramidase antibody.
Fig. 4
Fig. 4. Infection of human HSPCs from multiple sources
CD34+ cells from human umbilical cord blood, bone marrow and mobilized peripheral blood were transduced with LV/AC/huCD25. CD25 expression was assessed by flow cytometry using a PE-conjugated anti-CD25 antibody.
FIG. 5
FIG. 5. Transgene expression following direct LV delivery to neonatal mice
(A) One- to three-day-old neonatal animals were injected with LV/AC/huCD25 or LV/enGFP via the temporal vein. Plasma was collected from the PB at weeks 7, 10 and 14 post-viral delivery. The levels of sCD25 were measured by ELISA. Results are presented for each LV/AC/huCD25-treated mouse in the study. LV/enGFP and untreated mice showed no detectable levels of sCD25 (data not shown). (B) At 14 weeks post-viral delivery, mice were sacrificed and AC activity was measured in the organs. Shown are the results of liver enzyme activity. Values are represented as means ± SEM. For LV/AC/huCD25-treated and non-treated mice, n = 6; for LV/enGFP-treated mice, n = 7. Other organs showed no significant increase in AC activity over normal background levels (data not shown). * p < 0.05.
FIG. 5
FIG. 5. Transgene expression following direct LV delivery to neonatal mice
(A) One- to three-day-old neonatal animals were injected with LV/AC/huCD25 or LV/enGFP via the temporal vein. Plasma was collected from the PB at weeks 7, 10 and 14 post-viral delivery. The levels of sCD25 were measured by ELISA. Results are presented for each LV/AC/huCD25-treated mouse in the study. LV/enGFP and untreated mice showed no detectable levels of sCD25 (data not shown). (B) At 14 weeks post-viral delivery, mice were sacrificed and AC activity was measured in the organs. Shown are the results of liver enzyme activity. Values are represented as means ± SEM. For LV/AC/huCD25-treated and non-treated mice, n = 6; for LV/enGFP-treated mice, n = 7. Other organs showed no significant increase in AC activity over normal background levels (data not shown). * p < 0.05.
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