Prolonged Regulatable Expression of EPO from an HSV Vector Using the LAP2 Promoter Element

ORIGINAL ARTICLE Prolonged regulatable expression of EPO from an HSV vector using the LAP2 promoter element

Z Wu, M Mata and DJ Fink

We previously reported regulated expression of erythropoietin (EPO) over 4 weeks in the peripheral nerve in vivo, using a herpes simplex virus (HSV)-based vector containing a Tet-on regulatable gene expression cassette. To create a vector that would be appropriate for the treatment of chronic neuropathy, we constructed a HSV vector with expression of EPO under the control of the Tet-on system in which the HSV latency-associated promoter 2 element was used to drive the expression of the Tet-on transactivator. EPO expression from the vector was tightly controlled by administration of doxycycline (DOX) in vitro. One month after inoculation of the vector to transduce dorsal root ganglion (DRG) in vivo, administration of DOX-containing chow-induced expression of EPO. Mice with streptozotocin-induced diabetes, inoculated with the vector, were protected against the development of neuropathy by continuous administration of DOX-containing chow over the course of 3 months. Identical results were achieved when DOX was administered every other week over 3 months of diabetes, but administration of DOX, 1 week out of 3, provided only partial protection against the development of neuropathy. Taken together, these results suggest such a vector is well suited for clinical trial for the treatment of chronic or subacutely developing neuropathy.

Gene Therapy (2012) 19, 1107--1113; doi:10.1038/gt.2011.188; published online 17 November 2011 Keywords: HSV; diabetes; neuropathy; erythropoietin

INTRODUCTION expression of a neurotrophic factor might produce unanticipated Extensive preclinical animal studies indicate that systemic delivery effects requiring the discontinuation of treatment, it would of neurotrophic factors can prevent the development of be desirable to be able to regulate transgene expression. In a polyneuropathy,1--5 but translation of these findings into clinical proof-of-principle study, we previously reported construction and treatment has been hampered by off-target effects that preclude characterization of a non-replicating HSV-based vector in which a systemic administration of neurotrophic factors to patients in Tet-on system expressed under the control of the HCMV IEp was 6--10 used to regulate the expression of EPO in DRG neurons in vitro and doses that are required to produce effects in animal models. 17 To overcome this limitation, we developed a series of non- in vivo. EPO expression from the vector was strictly regulated by doxycycline (DOX), and intermittent expression of the transgene replicating herpes simplex virus (HSV)-based gene transfer vectors in vivo achieved by DOX administration 4 days out of 7 was to target and express neurotrophic factors to dorsal root ganglion sufficient to protect mice against the development of diabetes- (DRG) neurons. Transduction of DRG by subcutaneous inoculation induced neuropathy over a 4-week period.17 of replication-deficient HSV vectors expressing neurotrophin-3 or In the current study, we constructed a novel HSV vector in nerve growth factor prevents the progression of neuropathy 11,12 which the Tet-on system used to control EPO expression was caused by overdose of pyridoxine or exposure to cisplatin, and placed under the control of the HSV LAP2 element and tested HSV-mediated delivery of erythropoietin (EPO), neurotrophin-3, or the effect of regulated EPO expression from the vector on the vascular endothelial growth factor by subcutaneous inoculation development of diabetic neuropathy over a period of 3 months. protects against the development of sensory neuropathy in mice- We found that continuous expression of EPO achieved by 13 --15 rendered diabetic by streptozotocin (STZ). continuous administration of DOX to animals inoculated with The temporal course of transgene expression in DRG in vivo the vector preserved nerve function in rats with STZ-induced from HSV-based vectors is dependent on the promoter element diabetes. Intermittent expression of EPO achieved by administra- utilized. HSV vectors in which gene expression is driven by the tion of DOX containing chow every other week (1 week out of 2) human cytomegalovirus immediate-early promoter (HCMV IEp) provided a similar level of protection against the development produce relative short-term gene expression that persists for a of neuropathy. The results have important implications for the 11 period of weeks. HSV vectors in which transgene expression is development of HSV-based vectors to treat neuropathy. under the control of the HSV latency-associated promoter 2 (LAP2) element produce at least 6 months of biologically relevant transgene expression in both the central16 and peripheral11,14 RESULTS nervous systems. HSV LAP2-driven prolonged transgene expression would be Prolonged regulatable gene expression constructs appropriate for subacutely evolving chronic progressive condi- Prolonged regulatable gene expression was achieved by modify- tions like sensory polyneuropathy that evolves over a prolonged ing the commercially available two-component Tet-on system time frame. However, because of the possibility that prolonged consisting of a transactivator expression cassette and an inducible

Department of Neurology, University of Michigan and VA Ann Arbor Healthcare System, Ann Arbor, MI, USA. Correspondence: Dr DJ Fink, 1500 E Medical Center Drive, Ann Arbor, MI 48109, USA. E-mail: djfi[email protected] Received 19 August 2011; revised 19 October 2011; accepted 20 October 2011; published online 17 November 2011 Regulatable expression of EPO ZWuet al 1108 transgene expression element. We started with plasmid pSP27- were exposed to DOX (10 ng mlÀ1) for 2 days. Removal of DOX link--Tet-on that contains the expression cassette of the transacti- resulted in a rapid decrease in EPO concentration in the medium vator of the Tet-on system under the control of the HCMV IEp,17 from vL2rtEPO-infected cells and, by day 4 after DOX removal, no and replaced the HCMV IEp with the HSV LAP2 to create plasmid EPO was detectable in the medium (Figure 2c). Cells infected with pSP72-link-L2--Tet-on. The inducible EPO--HA (hemagglutinin) vL2rtEPO and continuously exposed to DOX, starting 2 days after and GFP (green fluorescent protein)--HA fusion expression infection, showed a substantial and statistically significant increase cassettes with expression of the transgene under control of TRE in EPO released into the medium measured on days 4--6. (tetracycline response element)-HCMV IEp were released from No detectable expression of EPO was seen in cells infected with plasmid pTRE-Tight-EPO--HA and pTRE-Tight-GFP--HA,17 and control vector vL2rtGFP and treated with DOX in a similar fashion ligated into the XhoI site of plasmid pSP72-link-L2--Tet-on to on either days 0 and 2, or days 4 through 6 (Figure 2d). create plasmids pSP72-TRE-Tight-EPO--HA-L2--Tet-on or pSP72- TRE-Tight-GFP--HA-L2--Tet-on. The regulatable gene expression vL2rtEPO expresses EPO under the control of DOX in vivo unit consisting of the transactivator expression element and the inducible transgene expression cassette was cleaved from plasmid To test the inducible expression of the transgene in vivo, mice pSP72-TRE-Tight-EPO--HA-L2--Tet-on or pSP72-TRE-Tight-GFP-- were inoculated subcutaneously in both hind feet with vL2rtEPO 8 m HA-L2--Tet-on and transferred to a shuttle plasmid SASB3, which (10 p.f.u. in 10 l PBS) and, 2 weeks later, rendered diabetic by IP contains sequences derived from HSV genome.17 The recombi- injection of STZ. One month after virus inoculation, normal chow nant vectors were generated by homologous recombination was replaced by a DOX-containing diet for 3, 4, 6 or 8 days, and between the end-point plasmid and the non-replicating HSV the amount of EPO in DRG determined by RT--PCR and ELISA. vector UL41E1G6 (for the EPO vector) or vEPO (for the GFP vector) Three-to-eight days of DOX administration induced the expression to produce the vector vL2rtEPO expressing the EPO--HA fusion of EPO mRNA (inset in Figure 3a) and protein (Figure 3a). No EPO peptide and the vector vL2rtGFP encoding for the GFP--HA fusion expression was detectable in the animals receiving the vector but peptide (Figure 1). not fed DOX-containing diet (day 0). To analyze the kinetics of the shut-off of transgene expression in the absence of DOX in vivo, animals were inoculated with vL2rtEPO and one month after virus vL2rtEPO expresses EPO under the control of DOX in vitro inoculation fed DOX-containing chow for 7 days, after which We tested the regulated expression of EPO from vL2rtEPO in animals were again fed normal chow. EPO mRNA (inset in response to exposure to DOX in complementing 7b cells. Vector Figure 3b) and protein (Figure 3b) levels in DRG, measured 2 and DNA replication, which is required for the HSV LAP2 to be active in 4 days after removal of DOX-containing chow fell to undetectable acute infections, occurs in those cells.18 7b cells were infected with levels by 4 days of normal chow. vL2rtEPO or vL2rtGFP at a multiplicity of infection (MOI) of 3 and DOX added 1 h after infection. EPO expression was assessed by À1 DOX-induced expression of EPO from the vector prevents the ELISA after 48 h of DOX treatment. 10 ng ml DOX resulted in progression of diabetic neuropathy in vivo release of 182 ng mlÀ1 EPO into the medium from cells infected To determine whether intermittent expression of EPO from the with vL2rtEPO, which did not increase significantly with increasing vector preserves nerve function in diabetic animals over a concentration of DOX up to 104 ng mlÀ1 (Figure 2a). However, prolonged time course, we tested the vector in a mouse model there was no detectable release of EPO from cells infected with of diabetic polyneuropathy. Male Swiss Webster mice were control vector vL2rtGFP and treated with DOX or from cells inoculated into the plantar surface of both hind feet with infected with vL2rtEPO but not exposed to DOX (Figure 2a). vL2rtEPO (108 p.f.u. in 10 ml PBS) and 2 weeks later rendered RT--PCR of RNA isolated from infected cells after 48 h of diabetic by intraperitoneal injection of STZ. Two weeks after STZ 10 ng mlÀ1 DOX treatment using EPO--HA specific primers showed injection (1 month after vector inoculation), diabetic animals an EPO-specific band in vL2rtEPO but not vL2rtGFP-infected cells infected with vector vL2rtEPO were tested with four different (Figure 2b). regimens: (1) normal food (no DOX); (2) DOX-containing chow 1 We examined the kinetics of EPO expression by measuring the week out of 3; (3) DOX-containing chow 1 week out of 2; or (4) amount of EPO released into the medium from infected cells using continuous administration of DOX-containing chow (Figure 4a). DOX on--off and off--on paradigms. 7b cells were infected with Diabetic animals with blood glucose concentration higher than vL2rtEPO or vL2rtGFP at an MOI of 0.01. A substantial release of 300 mg dlÀ1 were included in the study. As a positive control, EPO into the medium was observed, when vL2rtEPO-infected cells nondiabetic animals were fed normal chow. At 14 weeks of diabetes (16 weeks after vector injection, 3 months after initiation of DOX treatment), sensory nerve TRE K EPO HA pA pLAP2 K rtTA pA amplitude and velocity were determined, followed the next day by determination of thermal threshold, using the hot plate test.

TRE K GFP HA pA pLAP2 K rtTA pA Diabetic animals inoculated with vL2rtEPO and fed normal chow showed significantly reduced sensory nerve amplitude and conduction velocity consistent with the development of neuropathy (Figures 4b and c). Diabetic animals inoculated with expression cassette vL2rtEPO and fed DOX-containing chow continuously, or fed DOX- containing chow on a one-week on and one-week off schedule, Δ146207-151814 had amplitudes and conduction velocity indistinguishable from control, non-diabetic mice (Figures 4b and c). Diabetic animals Δ113322-114517 Δ126413-131930 inoculated with vL2rtEPO and fed DOX-containing chow 1 week out of 3 showed a marked decrease in sensory nerve amplitudes expression cassette and conduction velocity compared with naı¨ve animals, though Figure 1. Vector schematic. Two copies of the cassette, containing statistically significantly higher than that observed in diabetic the transactivator under the control of the HSV LAP2 promoter animals injected with the vector but not fed DOX-containing chow element and EPO under the control of a TRE, were inserted into the (Figures 4b and c). The behavioral results followed a similar ICP4 loci of the replication deficient parental HSV virus. rtTA: reverse pattern. Diabetic animals infected with vL2rtEPO and fed normal Tet-controlled transactivator. food had a significantly increased latency to withdraw from a

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DOX DOX Figure 2. Regulated expression of EPO from the vector by DOX in vitro. Complementing 7b cells were infected by vL2rtEPO or vL2rtGFP with a MOI of 3 and exposed to DOX of different concentrations. EPO concentration in the medium were measured by ELISA (a) and EPO mRNA levels from the 7b cells infected with vL2rtEPO or vL2rtGFP and cultured in the presence or the absence of 10 ng mlÀ1 DOX for 48 h determined by RT--PCR (b). To test the kinetics of the turn-on and turn-off of EPO expression from the vector, 7b cells were infected with vL2rtEPO or vL2rtGFP at a MOI of 0.01 and cultured either in 10 ng mlÀ1 DOX containing medium for 2 days and subsequently in normal medium for 4 days (c) or cultured in normal medium for the first two days after virus infection followed by 4-day culturing with 10 ng mlÀ1 DOX-containing medium (d). In each culture condition, a small volume of medium was collected every 2 days and EPO concentration in the medium measured by ELISA. painful thermal stimulus, but diabetic mice inoculated with progression of neuropathy resulting from chemotherapy, diabetes vL2rtEPO and fed DOX-containing food continuously, or fed or HIV infection.4,21 --23 In the previous work, we demonstrated that DOX-containing food every other week, had a normal withdrawal EPO expression in DRG achieved by subcutaneous inoculation of a latency that was identical to that of control, non-diabetic mice non-replicating HSV vector can be used to protect peripheral (Figure 4d). Animals inoculated with vL2rtEPO and fed DOX- nerve from damage in diabetic animals as demonstrated by containing food only 1 week out of 3 showed increased latency to reduced nerve fiber loss in the skin and increased expression of withdraw from a painful thermal stimulus relative to the control neuropeptide calcitonin gene-related peptide in the dorsal horn.15 naı¨ve animals. This result indicates that DOX-driven expression of We subsequently demonstrated that a non-replicating HSV vector, EPO only 1 week out of 3 is insufficient to protect the nerve in which EPO expression from a Tet-on-based HSV vector was against the effects of diabetes. controlled by administration of DOX, could be used to achieve similar results when DOX was administered 4 days out of 7 over the course of one month of diabetes.17 The major off-target effect DISCUSSION one might anticipate is using EPO to treat neuropathy would be The principal results of this study are that regulated expression of an increase in hematocrit, and, in our previous study, we found EPO from an HSV vector, using a Tet-on system in which the LAP2 that levels of HSV-mediated expression of EPO in DRG sufficient to element was used to drive expression of the Tet-responsive prevent neuropathy had no measurable effect on hematocrit.15 transactivator, provides prolonged regulated expression in the In this study, we replaced the HCMV IEp with the HSV LAP2 to peripheral nervous system, and that intermittent expression of drive the expression of the transactivator in the Tet-on system to EPO (every other week), achieved by intermittent administration achieve a long-term regulated transgene expression. In vitro and of DOX over a period of 3 months, is sufficient to protect against in vivo studies indicated that kinetics of the turn-on and turn-off of the development of neuropathy in diabetic animals. the expression of EPO from the vector by DOX was similar to that EPO, a hormone that primarily regulates red blood cell observed in the non-modified Tet-on-based EPO HSV vector in production, is a potent neuroprotective factor that can prevent our previous work.17 Moreover, we found intermittent expression neuron death either by inhibiting the production of reactive of EPO from the vector by feeding animals DOX-containing oxygen species or via PI3K/Akt/GSK-3b signaling.15,19,20 Adminis- chow, every other week, is sufficient to preserve nerve function in tration of EPO by intraperitoneal injection prevents the diabetic animals for at least 3 months.

& 2012 Macmillan Publishers Limited Gene Therapy (2012) 1107 --1113 Regulatable expression of EPO ZWuet al 1110 50 most promoters (including promoters considered constitutively active) placed into the HSV backbone are shut off over the course of a few weeks.11 We chose to use HSV LAP2 to drive expression of 40 the Tet-on system because (1) HSV LAP2 is naturally active for life in trigeminal and dorsal root ganglion neurons in humans and animals, once HSV establishes a latent state;35 and (2) animal 30 studies indicate that LAP2 can drive the long-term biologically relevant expression of therapeutic peptides in the central g protein) 11,14,16 μ and peripheral nerve systems. Neurotrophin-3 expressed 20 EPO from a HSV LAP2-based HSV vector prevented the progression of β-actin pyridoxine and diabetes-induced neuropathy at least for 6 11,14

EPO (pg/ months. HSV LAP2-driven expression of EPO and GDNF from 0 2468 10 a HSV vector protected dopaminergic neurons against MPTP and 6-OHDA-induced degeneration in mouse models of Parkinson’s disease for at least 6 months.16,36 0 Neurotrophic factors have been demonstrated to preserve 2648peripheral nerve function in animal models of many forms of 1--5 days of DOX administration neuropathy. Potentially, any one of a number of neurotrophic factors could be used as the transgene for developing a vector for 50 the treatment of neuropathy. In this proof of principle study, we chose to develop and test an EPO vector, but it is highly likely that similar results could be achieved with other neurotropic factors. 40 EPO There are no perfect animal models of human diabetic neuropathy. Therefore, we do not know in patients whether β -actin 1--week-on followed by 1-week-off of EPO expression will prove 0 2 4 30 sufﬁcient to prevent the progression of diabetic neuropathy clinically. However, even if continuous expression of EPO were g protein)

μ required to prevent progression of neuropathy in patients, 20 regulated expression would be desirable so that, in the event that side effects were observed, EPO expression could be stopped

EPO (pg/ by interrupting the administration of DOX. HSV-mediated gene 37 10 transfer to the DRG has moved into clinical trial and a phase-2 trial of HSV-mediated gene transfer for the treatment of pain is currently underway. The characterization of an HSV vector with 0 prolonged regulated gene expression has important implications 0 24 for the further development of this therapy to treat chronic problems like polyneuropathy. days after last DOX administration Figure 3. Induction of EPO expression from the vector by DOX in animals. To test the induction of EPO expression from vL2rtEPO MATERIALS AND METHODS in vivo, animals were subcutaneously inoculated with the vector and Cells and viruses fed DOX-containing diet for 3, 4, 6 or 8 days 1 month after vector inoculation, EPO mRNA (inset in a) and protein (a) levels in DRG 7b cells (Joseph Glorioso, University of Pittsburgh), a derivative of Vero measured by RT--PCR and ELISA. To test the shut-off of the cells that expresses HSV ICP27 and ICP4, were maintained and grown in expression of EPO from the vector in the absence of DOX, animals Dulbecco’s modified Eagle’s essential medium supplemented with 10% were inoculated with the vector and fed DOX-containing diet for 7 fetal bovine serum (Atlanta Biologics, Atlanta, GA, USA), 100 units per ml days followed by normal food for 2 or 4 days, EPO mRNA (inset in b) penicillin, 100 mg per ml streptomycin sulfate, 0.03% glutamine, and and protein (b) levels in DRG were measured by RT--PCR and ELISA. 0.375% sodium bicarbonate in a 5% CO2 atmosphere. The parental HSV vectors UL41E1G6 (provided by Joseph Glorioso, University of Pittsburgh) and vEPO (Zetang Wu et al., unpublished) used for generation of the recombinant viruses in this study are HSV-null mutants deleted for the We chose to use the Tet-regulated system for regulated essential gene ICP27 with both copies of the IE gene ICP4 replaced by GFP gene expression because (1) the inducer DOX is approved for or EPO. UL41E1G6 and vEPO were propagated in 7b cells and the virus titer human use; (2) a safe dose used in humans has been proven was determined by plaque assay.38 to be sufficient to induce transgene expression in rat, mice and nonhuman primates;24,25 (3) in vitro and in vivo studies demonstrate that transgene expression can be tightly regulated Construction of regulatable EPO--HA and GFP--HA expression by DOX;26 --28 and (4) there is no transactivator-specific immunity plasmids pSASB3-L2-EPO--HA and pSASB3-L2-GFP--HA produced in animals receiving a Tet-on-based transgene expres- To generate prolonged regulatable transgene expression plasmids, HCMV sion vector.29,30 We chose to use Tet-on system rather than Tet-off IEp promoter, which drives the expression of the transactivator in the Tet- system, because we reasoned that, in clinical use, should an on system, was replaced by the HSV LAP2 promoter. 600 bp HSV LAP2 was adverse off-target effect emerge, the patient would only need to amplified from replication-deficient HSV mutant UL41E1G6-infected 7b cell stop taking the activation drug to correct the problem.31 DNA by PCR using an upper primer, 50-CCG CTC GAG GGG TGG TGC GAA Regulation of transgene expression over an extended time AGA CTT TCC-30 and a lower primer, 50-CCG GAA TTC GAA GCA GGT GTC frame has been accomplished using a variety of different vector TAA CCT ACC TG-30. The PCR product was ligated into the XhoI and EcoRI platforms.32 --34 However, in contrast to the vectors employed in sites of plasmid pSP72-Link--Tet-on to replace the HCMV IEp promoter to the previous reports, in which prolonged expression of the drive the expression of the transactivator resulting in plasmid pSP 72-link- regulatable element could be achieved using a housekeeping L2--Tet-on. Plasmid pSP72-Link--Tet-on is a plasmid-modified from plasmid gene promoter32,34 or a tissue-specific constitutive promoter,33,34 Tet-on (Clontech, Mountain View, CA, USA) by changing the MCS to

Gene Therapy (2012) 1107 --1113 & 2012 Macmillan Publishers Limited Regulatable expression of EPO ZWuet al 1111 25

*** DOX dosing schedule 20 0 V) 1/3 μ 15 *** 1/2 continuous (c) 10 amplitude ( wks 5 STZ vector 0 0 1/3 1/2 c control diabetic

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0 0 0 1/3 1/2 c 0 1/3 1/2 c control diabetic control diabetic Figure 4. Protective effect of intermittent expression of EPO from the vector on the development of diabetic neuropathy. Animals were subcutaneously inoculated with vector vL2rtEPO and rendered diabetic by STZ injection 2 weeks after vector inoculation. Two weeks after STZ administration, diabetic animals receiving the vector were fed DOX-containing food at different schedules for 3 months. Nerve conduction studies and hot-plate testing were conducted at the end of the third month of treatment. (a) Schematic of the treatment protocol; (b) amplitude; (c) nerve conduction velocity; and (d) withdrawal latency from painful thermal stimulus in naı¨ve and vL2rtEPO-infected animals treated with DOX at different schedules. *Po0.005; **Po0.001; ***Po0.0001. facilitate the downstream manipulation of cloning, which constitutively with expression under the control of HCMV IEp were inserted into the ICP-4 expresses the transactivaor under the control of HCMV IEp.17 Regulatable loci of the HSV genome), was used for generation of the recombinant virus EPO--HA and GFP--HA fusion expression cassettes were released from allowing for green-white screening for the recombinant virus under plasmids pTRE-Tight-EPO--HA and pTRE-Tight-GFP--HA, which were con- fluorescence microscopy. structed in our previous work and express rat EPO and GFP--HA fusions in the presences of DOX and the transactivator under the control of the Animal experiments tetracycline-response element (TRE)-minimal HCMV IEp fusion promoter,17 Male Swiss Webster mice weighing 20--25 g (Charles River, Wilmington, and ligated into the XhoI site of plasmid pSP72-link-L2--Tet-on. The MA, USA) were used for all the experiments. All animal procedures in this resultant plasmids pSP72-TRE-Tight-EPO--HA-L2--Tet-on and pSP72-TRE- study were performed in compliance with approved institutional animal Tight-GFP --HA-L2--Tet-on express EPO and GFP--HA fusions in the care and use protocols. For analyzing regulation of EPO expression from presence of DOX. The whole EPO and GFG--HA fusion-inducible expression the vector by DOX, mice were inoculated with vL2rtEPO in both hind feet units were released from plasmid pSP72-TRE-Tight-EPO--HA-L2--Tet-on (108 plaque-forming units (p.f.u.) in 10 ml PBS) and 2 weeks later rendered and pSP72-TRE-Tight-GFP--HA-L2--Tet-on by BglII cleavage and cloned into diabetic by STZ injection twice within 2 days with a dose of 100 mg per kg the BamHI site of plasmid pSASB3 (Joseph Glorioso, University of animal weight. Two weeks after STZ injection, animals with blood glucose Pittsburgh) for facilitating the construction of HSV-based EPO and GFP concentration higher than 300 mg dlÀ1 were fed 625 mg kgÀ1 DOX- expression vectors. The resulting plasmids pSASB3-LAP2-EPO and pSASB3- containing diet (Harlan laboratory, Madison, WI, USA). Six animals were LAP2--GFP were used for generation of the corresponding recombinant euthanized at day 0, 3, 4, 6 and 8 after DOX treatment and L4-6 DRGs vectors. removed. EPO expression in DRG was measured by RT--PCR and ELISA. To examine whether removal of DOX treatment turns off the EPO expression Construction of long-term regulatable EPO and GFP expression from the vector induced by DOX in animals, animals were fed DOX- vectors containing chow for 7 days 1 month after virus infection and, after which, Recombinant HSV vector vL2rtEPO-expressing rat EPO--HA was generated animals fed normal food for 2 or 4 additional days, then euthanized and based on homologous recombination between plasmid pSASB3-LAP2-EPO L4-6 DRGs removed for EPO expression assays. To determine whether regulated expression of EPO from the vector preserves sensory nerve and replication-deficient HSV-1 genome DNA (UL41E1G6), in complementing 7b cells, and two copies of the regulatable EPO expression unit were function in diabetic animals, animals were infected by the vector vL2rtEPO inserted into the ICP4 loci of the HSV genome because the regulatable and rendered diabetic two weeks after virus injection. Diabetic animals gene expression unit in plasmid pSASB3-LAP2-EPO was flanked by were either fed normal food or fed DOX-containing chow at different schedules (continuously, 1 week out of 2 or 1 week every 3 weeks) sequences corresponding to the up- and down-stream sequences of the ICP4 open reading frame in HSV at the 50- and 30-ends, which serve as for 3 months (12 weeks). Sensory nerve amplitude and conduction velocity the basis for homologous recombination between the plasmid DNA and and the sensitivity of animals to a heat stimulus were measured at the end the viral genome. Generation of the recombinant EPO-expression vector, of the 12th week after DOX treatment. and the screening, purification and confirmation of the recombinant virus were conducted using the methods previously described.17 Recombinant Electrophysiological testing vector vL2rtGFP that expresses GFP --HA was created using similar methods Diabetic neuropathy was determined in these animals by abnormalities in utilized for the generation of vector vL2rtEPO except that parental virus vEPO peripheral nerve electrophysiology (amplitude, conduction velocity and (a replication-deficient recombinant HSV virus, in which two copies of EPO latency). Sensory nerve recordings were performed on the right hind foot

& 2012 Macmillan Publishers Limited Gene Therapy (2012) 1107 --1113 Regulatable expression of EPO ZWuet al 1112 using a Nicolet Viking III EMG device (Nicolet Biomedical, Madison, WI, USA). supported by NIH grants DK044935 and NS038850, and grants from the Department Mice were anesthetized with isoflurane for testing, and subcutaneous of Veterans Affairs and the Juvenile Diabetes Research Foundation. temperature maintained at 36--37 1C. The hind limbs were secured at an angle of 301 relative to the body and a ground electrode inserted into the tail, electrodes inserted into the sciatic notch as the recording electrode, whereas the stimulating electrode was placed at the ankle, and the REFERENCES reference electrode was positioned at the first digit.17 1 Hayakawa K, Itoh T, Niwa H, Mutoh T, Sobue G. 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The synthesized Protective effect of herpes simplex virus-mediated neurotrophin gene transfer in complementary DNA was used for quantification of mRNA levels by cisplatin neuropathy. Brain 2004; 127 (Part 4): 929 --939. semi- quantitative PCR. 13 Chattopadhyay M, Krisky D, Wolfe D, Glorioso JC, Mata M, Fink DJ. HSV-mediated gene transfer of vascular endothelial growth factor to dorsal root ganglia prevents diabetic neuropathy. Gene Therapy 2005; 12: 1377 --1384. Polymerase chain reaction 14 Chattopadhyay M, Mata M, Goss J, Wolfe D, Huang S, Glorioso JC et al. Prolonged Polymerase chain reaction (PCR) amplification was carried out in 50 ml using a preservation of nerve function in diabetic neuropathy in mice by herpes simplex standard protocol with an initial denaturing step at 94 1C for 5 min followed virus-mediated gene transfer. Diabetologia 2007; 50: 1550 --1558. by either 25 cycles for detection of the b-actin mRNA level or 30 cycles for 15 Chattopadhyay M, Walter C, Mata M, Fink DJ. Neuroprotective effect of herpes the measurement of the EPO--HA mRNA level at 951Cfor1min,601Cfor simplex virus-mediated gene transfer of erythropoietin in hyperglycemic dorsal 30 sec, 721C for 1 min and the products separated on a 1% agarose gel. root ganglion neurons. Brain 2009; 132 (Part 4): 879 --888. The primers used in PCRs are as follows: for identification of vL2rtEPO identity 16 Puskovic V, Wolfe D, Goss J, Huang S, Mata M, Glorioso JC et al. Prolonged biologically active transgene expression driven by HSV LAP2 in brain in vivo. or semi-quantification of exogenous EPO expression, the forward primer is Mol Ther 2004; 10:67--75. 0 0 5 -CCGGAATTCGCCAGGCGCGGAGATG-3 and the reverse primer that 17 Wu Z, Mata M, Fink DJ. Prevention of diabetic neuropathy by regulatable 0 contains EPO and HA sequences is 5 -CGGGATCCTCAAGCGTAATCTGGAACAT expression of HSV-mediated erythropoietin. Mol Ther 2011; 19: 310 --317. C-30. For identification of vL2rtGFP identity, the forward prime is 50-CGGAATT 18 Goins WF, Sternberg LR, Croen KD, Krause PR, Hendricks RL, Fink DJ et al. A novel CCGCCACCATGGCTAGCA AAGGAG-30 and the reverse primer is 50-CTAGATCA latency-active promoter is contained within the herpes simplex virus type 1 AGCGTAATCTG GAACATCGTATGGGTATGC-30. For semi-quantification of UL flanking repeats. J Virol 1994; 68: 2239 --2252. b-actin mRNA levels, the forward primer: 50-CAGTTCGCCATGGATGACGATA 19 Shuai H, Zhang J, Zhang J, Xie J, Zhang M, Yu Y et al. Erythropoietin protects TC-30, and the reverse primer: 50-CACGCTCGGTCAGGATCTTCATG-30. pancreatic beta-cell line NIT-1 cells against cytokine-induced apoptosis via phosphatidylinositol 3-kinase/Akt signaling. Endocr Res 2011; 36:25--34. 20 Dang J, Jia R, Tu Y, Xiao S, Ding G. Erythropoietin prevents reactive oxygen species Statistical analysis generation and renal tubular cell apoptosis at high glucose level. Biomed The statistical significance of the difference between groups was Pharmacother 2010; 64: 681 --685. determined by Student t-test and results expressed as means ±s.d. 21 Cervellini I, Bello E, Frapolli R, Porretta-Serapiglia C, Oggioni N, Canta A et al. The neuroprotective effect of erythropoietin in docetaxel-induced peripheral neuropathy causes no reduction of antitumor activity in 13762 adenocarcinoma- CONFLICT OF INTEREST bearing rats. Neurotox Res 2010; 18: 151 --160. 22 Schmidt RE, Green KG, Feng D, Dorsey DA, Parvin CA, Lee JM et al. Erythropoietin The authors declare no conflict of interest. and its carbamylated derivative prevent the development of experimental diabetic autonomic neuropathy in STZ-induced diabetic NOD-SCID mice. Exp Neurol 2008; 209: 161 --170. 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