Human hepatocytes used in viral correction experiments were derived from the discarded liver of a 5 year old boy with mut^o class methylmalonic acidemia undergoing a combined renal and hepatic transplant procedure. The patient initially presented with hyperammonemia and metabolic crisis in the presence of extreme methylmalonic acid elevations on the second day of life. Complementation and [1-¹⁴C] propionate incorporation studies on skin fibroblasts indicated a mut⁰ lesion. Subsequent sequencing of the MUT gene revealed two early nonsense mutations, E224X and R228X9. At the age of five, the patient underwent a combined liver kidney transplant from a deceased donor in which the whole organ liver and left kidney allografts were implanted in one procedure.

Mut and wild-type murine embryonic fibroblasts have been described2425 and were isolated after a timed mating between mice carrying a targeted deletion of the Mut gene.

Hepatocytes from the affected patient were isolated after the liver was removed as part of elective combined liver-kidney transplantation. The liver was considered a discarded surgical specimen because it was not suitable for transplantation and was donated by the family for research use. Patient studies were conducted in compliance with the Helsinki Declaration and were approved by the National Human Genome Research Institute Institutional Review Board as part of NIH study 04-HG-0127 "Clinical and Basic Investigations of Methylmalonic Acidemia and Related Disorders" after informed consent was obtained. Hepatocyte isolation from resected liver specimens was approved by the University of Pittsburg Institutional Review Board (IRB Number 0411142). Immediately upon surgical removal of the native liver the organ was flushed with ice-cold University of Wisconsin solution and shipped on wet ice to the University of Pittsburgh cell isolation facility established as part of the NIH-funded Liver Tissue Procurement and Distribution System. Hepatocytes were isolated from as described by Strom et al. 2627, with some minor changes described here. Briefly, cells were isolated from the entire liver by a 3-step collagenase perfusion protocol. Catheters were sewn in place into the 3 of the large hepatic veins and the liver was placed in a sterile plastic bag, and connected to a pump that delivered perfusate at approximately 80 mls/minute/catheter. The liver was sequentially perfused with 1 liter of calcium and magnesium-free HBSS (Cambrex, Walkersville, MD) containing EGTA (1 mM), 1 liter of HBSS without EGTA. Finally, 1 liter of Minimum Essential Medium Eagle (EMEM, Cambrex, Walkersville, MD) containing 250 mg collagenase (Type XI, Sigma, St. Louis, MO) and 50 mg of DNAase (Sigma, St. Louis, MO) was recirculated until the tissue was digested (approximately 29 minutes). Following digestion, tissue was placed in a sterile beaker, covered in ice-cold buffer EMEM and thoroughly chopped with a sterile scissors. Buffer and cells were decanted through sterile gauze covered funnels. Hepatocytes were enriched relative to nonparenchymal cells by 3 consecutive centrifugation steps at 75 × g for 5 minutes each. The final cell pellet was resuspended in hepatocyte maintenance medium (HMM, Cambrex, Walkersville, MD) and the viability was assessed by trypan blue exclusion. Visual inspection revealed greater than 95% parenchymal hepatocytes that were then plated on six well plates as described 26and allowed to attach for 4 hr, washed twice in serum-free media to remove dead and unattached cells and maintained thereafter by daily changes with serum-free HMM media. Control hepatocytes were isolated from a second donor, a 63 year old Male, not suspected to have primary liver disease or methylmalonic acidemia.

A murine methylmalonyl-CoA mutase cDNA that contained a consensus Kozak sequence and minimal untranslated regions was isolated from C57/BL6 liver RNA after RT-PCR, sequenced and tested for enzymatic activity after expression in yeast using the succinate-thiokinase linked assay25. The gene was then cloned as an EcoRI fragment into the polylinker of pShuttle between the CMV promoter and the Sv40 polyadenylation signal (ViraQuest Inc., North Liberty, IA). This E1 shuttle expressing Mut from the CMV promoter was then used in the RAPAd^® (U.S. Patent #6,830,920) adenovirus construction system with an RSV eGFP expressing E3 backbone28. The dual-expressing virus was derived by recombination after co-transfection of the E1 shuttle and backbone into HEK293 cells. 7–10 days after the appearance of viral foci, the plates were harvested and amplified, and then virus particles were isolated by two rounds of CsCl gradients and dialyzed against storage buffer (ViraQuest Inc., North Liberty, IA). Vector genomes and plaque forming units (PFU) were measured in the final adenoviral preparations. Animal studies were reviewed and approved by the National Human Genome Research Institute Animal User Committee. The cell culture correction experiments used virus at a multiplicity of infection (MOI) of 1000. Direct intrahepatic injections of 1.0 × 10¹⁰ particles (3.3 × 10⁸ PFU) of the adenovirus in mixed background (C57Bl6xSv129Ev) neonatal mice were accomplished using a 32 gauge Hamilton syringe. The animals were sacrificed 4 days later for dissection and analysis of eGFP expression.

Methylmalonyl-CoA mutase activity was determined by measuring [1-¹⁴C] propionate incorporation into macromolecules as described29. All propionate incorporation assays were performed in triplicate. [1-¹⁴C] Sodium propionate was purchased (Perkin Elmer, Boston, MA) as a custom preparation at a specific activity of 55.0 mCi/mmol (2 mCi/ml).

Whole cell extracts from the cell lines were analyzed by immunoblotting and probed with affinity purified, rabbit polyclonal antisera raised against the murine methylmalonyl-CoA mutase enzyme or beta-actin (Abcam, Cambridge, MA). The anti-mutase antibody was used at a dilution of 1:750, and beta-actin was used at 1:5,000. Goat anti-rabbit (Chemicon, Temecula, CA) was used as a secondary antibody at a dilution of 1:10,000 with chemiluminescent detection (Pierce Biotechnology, Rockford, IL). Recombinant murine methylmalonyl-CoA mutase enzyme was used as a positive control in Western blot analysis experiments. Human liver extracts were prepared from an anonymous liver donor not known to have methylmalonic acidemia or the mut⁰ patient described above.

Transfection and recombination in HEK293 cells using the RAPAd^® adenovirus method was used to produce replication deficient adenovirus particles that express the murine methylmalonyl-CoA mutase gene in the E1 region and eGFP from the E3 region28. The resulting replication deficient adenovirus expresses both genes from two independent promoters, Mut from the CMV and eGFP from the RSV (Figure 1). The virus was expanded, amplified and concentrated to an infectious titer of 1.2 × 10¹² particles/ml (corresponding to 4.0 × 10¹⁰ PFU/ml), indicating its stability during growth and replication. The strong viral promoters were selected to promote high-level expression in a wide spectrum of tissues types, in vivo and in vitro.

The bifunctional adenovirus was delivered by direct hepatic injection into the liver of one day old wild-type mice (N = 8). A total of 1.2 × 10¹⁰ (3.3 × 10⁸ PFU) adenoviral particles were administered in a total volume of 10 microliters. Four days post injection the animals were sacrificed and the organs were inspected for fluorescence using a dissecting microscope equipped with a GFP filter. The GFP expression was easily visualized, with a higher concentration of signal seen in the lobe that was likely the direct target of injection (Figure 2A). The presence of green fluorescence, not observed in the uninjected mice, indicates that the RSV-eGFP promoter functioned in vivo. Other organs, such as the heart and kidney, lacked signal and GFP expression was not observed in the uninjected mice. None of the wild type mice injected at this dose perished, suggesting that the virus and procedure were well tolerated.

Mut MEFs derived from methylmalonyl-CoA mutase knock-out mice were used to determine the efficacy of gene correction from the virus24. Methylmalonyl-CoA mutase protein and mRNA are not detectable in these cells (Figure 3 Lane 4; Chandler et al, submitted). The virus was incubated at a multiplicity of infection of 1000 for 16 hours. The cells were washed and then used to determine the extent of correction by Western blotting 72 hours post transduction. In parallel, enzymatic function was measured using macromolecular [1-¹⁴C] propionate incorporation. A wild-type murine fibroblast cell line served as a positive control in these experiments. Previous studies on the same cells using an E1 replaced, eGFP expressing adenovirus at the same MOI showed no effect on [1-¹⁴C] propionate incorporation and no gross cellular toxicity (Chandler et al, data not presented).

After correction, expression at the protein level was completely restored in the knock-out MEFs that had been infected. Figure 3A (Lane 4) shows that the Mut cells make no detectable methylmalonyl-CoA mutase protein compared to the wild type control (Lane 3), which has a single band migrating at 78 kDa. This band corresponds to the expected size of the murine enzyme after processing and is the same size as recombinant murine methylmalonyl-CoA mutase (Figure 3A, Lane 2). When normalized to actin, the enzymatic expression level in infected cells appears increased over wild type cells (compare Figure 3A Lane 5 to Lane 3).

We also measured the activity of the methylmalonyl-CoA mutase enzyme by evaluating propionate metabolism in Mut fibroblasts and compared it to Mut MEFs exposed to the virus. As demonstrated in Figure 3B, the [1-¹⁴C] propionate incorporation in the transduced cells nearly corrects to the levels observed in wild type cells, demonstrating that viral directed expression provides functional enzymatic correction.

The native liver from a patient with mut^o methylmalonic acidemia was harvested as part of a combined liver-kidney transplantation. Hepatocytes were isolated using collagenase perfusion. Cells were plated at a density of 5 × 10⁵ per well on collagen-coated 6-well plates (Falcon) and appeared large, hexagonal and healthy. Control hepatocytes, derived from an 63 year old anonymous male donor, were prepared in an identical fashion and were used to measure native [1-¹⁴C] propionate in an independent experiment.

The hepatocytes were infected at a MOI of 1000, as previously described. Twenty-four hours after infection the cells exhibited uniform GFP expression (Figure 2B). To access viral mediated correction, functional studies were performed on the hepatocytes with and without exposure to the adenovirus for 48 h. Compared to a control human liver extract (Figure 4A, lane 1), extracts derived from the human mut^o liver (Figure 4A, lane 2) and uninfected hepatocytes (Figure 4A, lane 3) revealed no cross reactive material even after prolonged exposure of the Western blot. Genetic studies on this patient previously identified two nonsense mutations (E224X and R228X)9 and these experiments confirmed that there was no read through. Post-infection, 10 micrograms of the mut^o corrected hepatocyte extract exhibited a large increase in immunoreactive enzyme as assessed by Western blotting (Figure 4A, Lane 4). When compared to the control human liver extract (Figure 4A, Lane 1), which contained 20 micrograms of protein, the corrected cells (Figure 4A, Lane 4) have a substantially larger band, suggesting an increased capacity in the virally infected cells for expression of the methylmalonyl-CoA mutase enzyme, even if normalization to actin is not considered in the comparison.

Enzymatic studies revealed a greatly increased metabolic capacity of the mutant hepatocytes after viral correction (Figure 4B). [1-¹⁴C] propionate incorporation in the virus exposed mut⁰ hepatocytes increased to approximately 20 nmol/mg/18 h (a factor of 7) over the uninfected cell line and exceeded the activity of control primary hepatocytes (Figure 4B). It should be noted that the control hepatocytes were derived from an older patient and hence may have a reduced propionate capacity compared with what might be observed in hepatocytes from a child, because of issues such as growth rate and/or other age related phenomenon. Unfortunately, an age and sex matched liver was not available for similar studies. The greatly increased [1-¹⁴C] propionate incorporation displayed by the mut^o cell line after viral correction indicates that hepatocytes have a large capacity for propionate metabolism and is consistent with the robust expression observed in the Western studies. In all the adenoviral delivery experiments, the infected cells appeared morphologically normal at the time enzymatic and Western studies were performed.