Design and packaging of lentiviral vectors

feEPO mRNA was previously PCR amplified from feline renal tissue–derived RNA, and the resulting reverse-transcribed cDNA was incorporated into an initial lentiviral vector system, referred to here as Vector A.²⁹ The feEPO gene (cDNA) was subsequently utilized in the design and construction of a novel third-generation, replication-defective lentiviral vector, referred to here as Vector D. A UC Davis Biological Use Authorization (BUA; #895) was approved for the use of this vector. The plasmid-encoded feEPO cDNA was confirmed by sequencing (GenBank JQ413414). Vector D features an HIV-derived and gene-deleted backbone with an early promoter of simian virus 40 (SV40). The SV40 promoter (Vector D; Figure 1) was selected as a constitutively active promoter with moderately attenuated function relative to the previously utilized cytomegalovirus (CMV) promoter.³⁵

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Vector D construct schematic. CMV = Cytomegalovirus. EPO = Erythropoietin. feEPO = Feline EPO. HSV TK = Herpes simplex virus thymidine kinase. IRES = Internal ribosome entry site. Puro = Puromycin-N-acetyltransferase gene. RRE = rev response elements. RSV = respiratory syncitial virus. SV40 = Simian virus 40.

Citation: American Journal of Veterinary Research 85, 6; 10.2460/ajvr.23.12.0280

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The SV40 promoter was situated directly 5’ to the cloned feEPO gene (cDNA), which was linked through an internal ribosome entry site to the herpes simplex virus thymidine kinase (HSV-TK) gene (suicide gene).³⁶ The inclusion of the thymidine kinase gene hypothetically enables the pharmacologically mediated and selective elimination of the vector-transduced cells. In the event of an adverse supraphysiologic response to the feEPO transgene (excessively elevated PCV), the suicide gene product (HSV-TK) phosphorylates the nontoxic prodrug ganciclovir (GCV) into the cytotoxic metabolite GCV triphosphate. GCV triphosphate is subsequently incorporated into host cell genomic DNA during cell replication, thereby inhibiting DNA synthesis and resulting in apoptosis of the transduced cell.³⁷ Selective elimination of transduced cells requires cell replication.

A control vector was also designed based on the HIV-derived backbone and featured the EF1A promoter regulating expression of the reporter gene mCherry (Figure 1). The control vector also featured a second internal promoter (CMV) regulating expression of the enhanced green fluorescent protein (GFP) and an antibiotic resistance gene (puromycin-N-acetyltransferase gene) linked to the enhanced GFP through an autocleaving 2A site.³⁸ The plasmid constructs for each vector were assembled by VectorBuilder (VectorBuilder.com) and were confirmed by sequencing.

Infectious lentiviral vectors were packaged and titered by VectorBuilder as transducing units per mL (TU/mL). Ultrapurified lentiviral vectors were shipped frozen from VectorBuilder on dry ice to Davis, California in Hanks’ balanced salt solution buffer, with titers ranging from 8.5 X 10⁸ and 1.9 X 10⁹ TU/mL.

Detection of feEPO protein using western blot assays

A series of western blot (WB) assays were performed to identify the presence of feEPO protein in tissue culture cells and/or culture supernatants. For each WB, controls included 5 μL (10 U) of human recombinant EPO protein (epoetin alfa [EPOGEN], positive control) and lysates from nontransfected/nontransduced cells (negative control).

Either cell lysates, culture supernatants, or both were collected for WB analyses. Cultured cells were detached from the plate with 0.25% Trypsin-EDTA (Gibco), washed with PBS (Gibco), centrifuged at 500 X g for 5 minutes, and resuspended in lysis buffer (150 mM NaCl, 50 mM Tris base, 1% NP-40, 0.25% deoxycolic acid, 0.1% SDS, pH 7.4) and protease inhibitor complex (Thermo Scientific). Cell lysate samples were then incubated on ice for 30 minutes and centrifuged at 16,000 X g for 10 minutes, and the cleared extract was transferred to a new tube. Culture supernatants were centrifuged at 500 X g for 5 minutes, transferred to a clean tube, and centrifuged at 3000 X g for 5 minutes before storage at –20 °C prior to WB analysis.

In preparation for electrophoresis, 13 µL of extracted protein or processed supernatant was mixed with 5 µL of 4X LDS Sample Buffer RunBlue (Abcam) and 2 µL of 10X DTT Reducer RunBlue (Abcam). Lysate samples were incubated for 10 minutes at 70 °C. The molecular weight marker, cell lysates, culture supernatant samples, and positive and negative control samples were electrophoresed on a 4% to 20% SurePAGE, Bis-Tris polyacrylamide gel (GenScript) at 150 V for 50 minutes using a PowerPack 200 Universal Power Supply (BioRad) and then transferred to a polyvinylidene fluoride (PVDF) membrane (BioRad) using a Mini-PROTEAN Tetra electrophoresis system (BioRad) at 5 °C for 12 hours at 50 V with constant stirring. The PVDF membrane was washed 3 times with Tris-buffered saline 0.1% Tween-20 (TBS-T) and placed in blocking buffer consisting of TBS-T 5% nonfat dry milk for 1 hour at room temperature. The PVDF membrane was washed 3 times with TBS-T before incubation with rabbit anti-human EPO antibody (Bio Rad) at a dilution of 1:1,000, rocking overnight at 5 °C. The membrane was subsequently washed 3 times with 10 mL of TBS-T prior to incubation with a goat anti-rabbit IgG-HRP Immunopure antibody (Thermo Fisher Scientific) at a dilution of 1:10,000 for 1 hour at room temperature. Antibodies were diluted in TBS-T with 5% bovine serum albumin.

In vitro experiments

An in vitro experiment was performed to evaluate the Vector D SV40 promoter-regulated expression of feEPO and to assess the functionality of the HSV-TK suicide gene system. Crandell-Rees Feline Kidney (CRFK) cells were cultured in Dulbecco’s modified Eagle medium/10% FBS in 12-well tissue culture plates (Corning) at a concentration of 4.5 X 10⁵ cells/well and incubated at 37 °C and 5% CO₂. Experimental groups included 1) nontransduced CRFK control cells, 2) CRFK cells transduced with the control vector lentivirus (multiplicity of infection [MOI] of 10), 3) CRFK cells transduced with Vector D lentivirus (MOI of 10), and 4) CRFK cells transduced with Vector D lentivirus (MOI of 10) and subsequently treated with GCV (ThermoFisher) at a concentration of 25 µM 24 hours post-transduction for 3 consecutive days. The concentration of GCV utilized was based on the determination of cytotoxicity for GCV used alone with CRFK cells, which was not observed at 25 µM (50% cytotoxicity concentration determined to be 52.3 µM using CellTox Green Cytotoxicity assay per the manufacturer’s protocol; Supplementary Figure S1). All experimental conditions were carried out in triplicate wells. Cells were transduced 24 hours after plating the CRFK cells by aspirating off the culture media and replacing it with the appropriate amount of Vector D or control vector lentivirus diluted in 2 mL fresh media.

An inverted fluorescent microscope (EVOS digital inverted microscope and cell imaging system; Life Technologies) was used to visualize cell morphology at 24, 48, 72, and 96 hours post-transduction using bright-field illumination. Visual assessment was performed to assess cytotoxicity in the wells treated with GCV. For wells transduced with the control vector, GFP and mCherry fluorescence was used to confirm lentiviral transduction of CRFK cells qualitatively.

At each time point (24, 48, 72, and 96 hours post-transduction), supernatants were aspirated from the wells and centrifuged at 500 X g for 5 minutes, transferred to a new tube, and centrifuged at 3,000 X g for 5 minutes before storage at –20 °C prior to determination of feEPO protein production via WB analyses as previously described. All WB analyses utilized the same volume culture supernatant from wells seeded with a constant cell density.

In vivo rodent experiments (overview)

In order to demonstrate the in vivo efficacy, longevity of effect, and safety of the feEPO lentiviral gene therapy system, a series of rodent studies were performed. An initial pilot study using Fischer rats was followed by a series of lentiviral dose-escalation studies utilizing genetically modified EPO-deficient (genetically disrupted EPO gene) anemic mice. For these experiments, packaged, concentrated, and ultrapurified Vector D (1.9 X 10⁹ TU/mL) and control vector (8.5 X 10⁸ TU/mL) lentivirus were obtained from VectorBuilder.

Rat study

A pilot rat study was performed in order to initially demonstrate the in vivo efficacy of Vector D (UC Davis IACUC #19202; BUA #R2033). Eight 15-week-old female Fischer rats (Charles River) between 147 and 183 g in weight were randomized into 3 experimental groups: (Group A) Vector D plasmid DNA treated (n = 3), (Group B) inoculated with Vector D lentivirus (n = 3), and (Group C) untreated control rats (n = 2). Group A rats were injected IM into the hamstring muscles of the right caudal thigh with 100 ug sterile Vector D plasmid DNA diluted in Tris EDTA buffer (70 μL). Group B rats were each injected with 6.32 X 10⁵ TU Vector D divided into 2 injection sites (180 μL divided between right and left caudal thigh). The remaining 2 rats (Group C) served as untreated controls.

Using either a sterile #10 scalpel blade or a lancet (Medipoint) to create a small nick in the tail tip, peripheral blood samples were obtained once per week and loaded into 1 or 2 heparinized capillary tubes (Fisher Scientific). For each peripheral blood sample, the red blood cell PCV was determined by centrifuging the capillary tubes at 12,000 X g for 5 minutes (Beckman Microfuge 11) and determining the percentage of packed red blood cells in the total blood volume (PCV). The peripheral PCV was determined for each rat for the 2 weeks prior to treatment in order to establish baseline values. The peripheral PCV and body weights were assessed on a weekly basis for each animal over a period of 12 weeks postinoculation. All of the in vivo experimental manipulations were approved and in alignment with the UC Davis Office of Research and IACUC.

At the 12-week terminus of the experiment, the rats were euthanized by isoflurane anesthesia followed by CO₂ asphyxiation and whole-blood exsanguination via cardiocentesis (no anticoagulant; approximately 3 to 5 mL blood obtained per rat). The terminal blood sample was allowed to clot and the serum was separated and frozen at –80 °C. Unclotted terminal whole blood was also collected into heparinized capillary tubes and utilized for a final PCV determination. A complete necropsy was performed for each rat, with tissues collected and preserved in 10% neutral-buffered formalin (NBF). Targeted fresh tissue samples (spleen, liver, kidney, and skeletal muscle of the caudal thigh) were frozen and archived in RNALater (Ambion) at –80 °C. Formalin-fixed tissues were fixed for 24 to 72 hours, routinely sectioned, embedded into paraffin blocks using standard protocols, stained with H&E, and examined microscopically by a board-certified veterinary anatomic pathologist (SEC).

Genetically modified anemic mouse studies

A series of in vivo studies using an EPO-deficient mouse model was approved and carried out under the UC Davis IACUC protocol #20925 and a BUA (R2033). A mixed colony of wild-type (WT), heterozygous (Het), and homozygous (Hom) genetically EPO-deficient mice (EPO-tag^h) was obtained as a gift of Dr. J. Solnick (UC Davis), originally sourced from France (N. Voituron).³⁹ The EPO-tag^h mice have the gene encoding the SV40 Large T antigen integrated into and disrupting the regulatory sequence of the endogenous murine EPO, gene resulting in targeted disruption of murine EPO expression and subsequent chronic anemia.^39,40 Mice were maintained in a vivarium managed by the UC Davis Teaching and Research Animal Care Services, where the mice were bred, in order to produce the required number of EPO-deficient mice (Hom EPO disruption) for the 2 murine studies detailed below. Founder mice were genotyped using PCR by the Solnick laboratory, who provided 15 mice total. The mice were genotyped by obtaining an approximately 1- to 2-mm long tail tip between 19 and 21 days of age for DNA isolation using the QIAamp DNA mini kit (Qiagen) following the manufacturer’s protocol for DNA purification from tissues. Isolated DNA samples were stored in microcentrifuge tubes at –20 °C prior to PCR analysis.

A set of 3 primers was utilized for genotyping the EPO-deficient anemic mice, facilitating the differentiation of WT, Het, and Hom EPO-disrupted animals. PCR was carried out using Applied Biosystems’ QuantStudio 3 Real-Time PCR System and PowerUp SYBR Green Master Mix following the manufacturer’s protocol for a 10-µL reaction. The 3 genotyping primers were used in a set of 2 real-time PCR reactions, and each reaction included a water template as a negative control and positive controls (DNA controls from previously genotyped WT, Het, and Hom EPO-disrupted mice). WT murine EPO primers included mEPO forward – 5'-CGC ACA CAC AGC TTC ACC C-3' and mEPO reverse – 5'-CTG TAG GGC CAG ATC ACC-3'. The primers for the EPO-disrupted gene included the mEPO forward primer paired with the SV40T reverse primer – 5'-GCC TAG GCC TCC AAA AAA GC. The WT PCR cycling conditions were as follows: 50 °C for 2 minutes and 95 °C for 2 minutes, followed by 40 cycles of 95 °C for 15 seconds, 56 °C for 15 seconds, and 72 °C for 15 seconds. The EPO-disrupted PCR cycling conditions were as follows: 50 °C for 2 minutes and 95 °C for 2 minutes, followed by 40 cycles of 95 °C for 15 seconds, 63 °C for 15 s, 72 °C for 15 seconds. The final step for both reactions included a dissociation curve to evaluate the specificity of primer binding. PCR amplicons were visualized using ethidium bromide–stained agarose gel electrophoresis. Expected amplicon sizes for mice with the WT EPO amplicon were 228 bp and 200 bp for mice with the EPO-disrupted genotype (SV40-mutant amplicon). Het mice possessing both a WT murine EPO allele and an EPO-disrupted allele were expected to exhibit both amplicons.

The mice were phenotyped using their tail blood to determine blood PCV. WT mice have a PCV of approximately 55%, Het mice approximately 40%, and Hom EPO-tag^h mice approximately 20%.⁴¹ Two of the founder mice were determined to be WT (lacking a disrupted EPO gene), 6 were Hom EPO deficient (EPO-tag^h), and 7 of the mice were Het for the disrupted EPO gene.

The initial murine study (murine study #1) was performed to evaluate the efficacy of feEPO transduction and longevity of effect when inoculated with a high dose of Vector D lentivirus (vector:body weight ratio relative to the rat study). All of the mice were approximately 13 weeks old at the initiation of the experiment. Twenty-four genetically confirmed Hom EPO-deficient mice with an average PCV of approximately 20% were divided into 2 groups: Group A mice were inoculated IM with Vector D lentivirus (16 mice; 10 males and 6 females), and Group B mice were inoculated IM with control vector lentivirus (8 mice; 5 female and 3 males). Group A mice each received 7.75 X 10⁷ TU Vector D in a total volume of 50 μL diluted with sterile PBS delivered into the right hamstring musculature. Group B mice each received 8.5 X 10⁶ TU control vector diluted with sterile PBS in a total volume of 50 μL, also delivered into the hamstring musculature of the right thigh.

Mouse behavior and body weight were serially assessed, peripheral blood was collected from the tail tip into heparinized hematocrit tubes and centrifuged at 12,000 X g for 5 minutes (Beckman Microfuge 11), and a peripheral blood PCV was determined for each animal on a weekly basis. At the end of the 9-week study, the mice were euthanized using isoflurane anesthesia followed by CO₂ asphyxiation and cardiac exsanguination (approx 0.5 to 1 mL per mouse). Terminal blood was used to determine PCV, and serum was separated by centrifugation and serum stored at –80 °C. A complete necropsy was performed for all animals, and tissues were preserved in 10% NBF. For select tissues (caudal thigh skeletal muscle, spleen, liver, and kidney), approximately 5 to 15 mg of each were collected fresh in 700 µL RNAlater solution (Ambion), and a duplicate set of tissues was archived at –80 °C. Tissues collected in formalin were fixed for a minimum of 24 hours prior to trimming into cassettes, embedded in paraffin wax, cut into 4-µM sections, mounted on glass slides, and stained with H&E for histologic evaluation. For bone marrow evaluation, both right and left femora were fixed in 10% NBF for a minimum of 24 hours and then decalcified in 15% formic acid for approximately 48 to 72 hours prior to processing for histologic evaluation as described above. All murine tissues were examined by a board-certified veterinary anatomic pathologist (S.E.C.).

In the second murine experiment (murine experiment #2), the inoculating dose of Vector D lentivirus was increased to 1 X 10⁸ TU/mouse, one group of mice received a second dose of the lentiviral vector (same amount as initial), and the IP administration of GCV was assessed for in vivo toxicity in transduced mice. Since the transduced skeletal myocytes are not a replicating cell population, we did not anticipate a GCV-induced termination of the gene therapy effect (eg, A reduction in PCV). This murine study was approved under the UC Davis IACUC #22226 and BUA R2033.

Fifteen genetically confirmed EPO-deficient anemic mice were randomly divided into 3 groups of 5 animals each: Group A control mice were inoculated with control vector lentivirus (2 females, 3 males), Group B mice were inoculated with 2 serial doses of Vector D lentivirus (2 females, 3 males), and Group C mice were inoculated with a single dose of Vector D lentivirus followed by GCV treatment (2 males, 3 females).

The inoculating doses of both the control vector and Vector D lentiviruses were increased to 1 X 10⁸ TU/mouse and administered IM in the same manner and location as the first murine study, with Group A control mice receiving the control vector diluted in sterile PBS in a total volume of 39 μL and Group B and C mice receiving Vector D in a volume of 53 μL. Peripheral blood was obtained from the tail tip, and was PCV determined along with body weight on a weekly basis. Each Group B mouse was reinoculated with a second dose of Vector D lentivirus (1 X 10⁸ TU/mouse) when an individual animal’s PCV fell below 25%. Based on published protocols,⁴² Group C mice received 50 mg/kg of GCV (Hospira; reconstituted with sterile water) beginning 3 weeks after inoculation with Vector D for 5 consecutive days via the IP route. At the time of GCV treatment in Group C mice, the PCV was determined to be elevated relative to the control animals and averaged 47.4% for the treated cohort. Reinoculated Group B mice were euthanized at 5 weeks postreinoculation. Group C mice were euthanized when their PCV values dropped below 25%, aside from 1 mouse, which was maintained to 31 weeks postinoculation (PCV of 28% at the time of euthanasia). Mice were euthanized using isoflurane anesthesia followed by CO₂ asphyxiation and cardiac exsanguination when their PCV had returned to near baseline (PCV < 25%). A complete necropsy, tissue collection, and histologic examination were performed for each mouse as described for the first murine study.

Statistical analysis

For the in vivo studies where there were more than 2 treatment groups involved, a 1-way ANOVA was performed for each dataset (time point) in order to identify significant differences between the 3 treatment groups, followed by Tukey multiple comparisons test. Brown-Forsythe tests were performed to confirm that group variances were equal and that, therefore, the variance assumption for a 1-way ANOVA test was valid. When only 2 experimental groups were present, a Student t test or Welch t test was performed for each dataset time point. Significance was defined as P ≤ .05.

Vector D transduction of feline CRFK cells

The efficient transduction of feline CRFK cells by the control vector (MOI 10) was confirmed using immunofluorescent microscopy. CRFK cells exhibited cytoplasmic GFP immunofluorescence in approximately 80% to 90% of the cells, subjectively, confirming cell transduction (Figure 2; data for mCherry not shown). Visual evaluation of CRFK cells at 96 hours post Vector D transduction and treated with GCV (bottom right of Figure 2) demonstrates morphologic evidence of cellular injury seen as rounding and occasional detachment of cells. These findings suggest that the CRFK cells were effectively transduced with Vector D lentivirus and that the HSV-TK gene product converted GCV into its toxic metabolite (functional suicide gene system in transduced replicating cells). Transduced CRFK cells that were not treated with GCV at the same time point do not demonstrate morphologic evidence of cytotoxicity (uniform monolayer of spindle-cohesive cells, upper left and lower left of Figure 2).

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Vector D transduction (MOI 10) of Crandell-Rees Feline Kidney (CRFK) cells. A—Representative images of nontransduced CRFK cells and those transduced with Vector D lentivirus with or without ganciclovir (GCV) treatment 96 hours post-transduction. B—Western blot image showing EPO protein detection from supernatants from the transduced CRFK cells. An appropriately sized band detected for the positive control (EPOGEN), and no bands detected for the Vector Control and nontransduced CRFK cells. GFP = Green fluorescent protein.

Citation: American Journal of Veterinary Research 85, 6; 10.2460/ajvr.23.12.0280

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In a WB assay performed with the treated cell-derived samples in parallel, supernatants from CRFK cells transduced with Vector D lentivirus demonstrate progressively increased production of feEPO protein over time (24 to 96 hours post-transduction), which becomes markedly attenuated with GCV treatment (Figure 2). EPO protein was not detected in CRFK cells transduced with the control vector (Vector) nor was it detected in the nontransduced CRFK cells (CRFK). An appropriately sized band was detected for the EPO-positive control lane (approx 43 kDa). All WB analyses were derived from the same volume of supernatant from wells seeded with a constant cell-seeding density to control for protein amounts.

Rats treated with Vector D lentivirus have a significant but transient increase in blood PCV

Fischer rats inoculated IM with Vector D lentivirus demonstrated a significant increase in blood PCV relative to uninoculated control animals and rats treated with Vector D plasmid DNA (Figure 3). The effect on PCV was statistically significant relative to the 2 other groups but was transient, persisting for only 2 weeks postinoculation. The mean ± SD PCV values at 1 and 2 weeks post-treatment were 52.3 ± 1.1, 59.2 ± 2.8, and 50.3 ± 2.9% for the uninoculated control, Vector D lentivirus treated, and plasmid DNA–treated groups at 1 week post-treatment and 51.3 ± 1.1, 58.0 ± 2.6, and 52.3 ± 2.8% for week 2 post-treatment, respectively. These results provide evidence that inoculation with Vector D lentivirus results in a transient increase in PCV in normal rats in vivo. After the 2-week time point, there were no significant differences in PCV detected between the 3 groups. No significant changes in behavior or weight gain/loss were identified in any of the study animals, although 1 rat in the plasmid-treated group died prematurely for an undetermined reason 2 days prior to the termination of the experiment.

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Mean packed cell volume (PCV) values versus time for the different rat treatment groups (error bars ± SD). A significant difference between the treatment groups is identified at 1 and 2 weeks post-treatment (ANOVA; P = .026). A Tukey multiple comparisons test identified a significant difference in the mean PCV values between the Vector D treatment group and the plasmid treatment group (P = .03) and no statistical significance when comparing the control group against the Vector D plasmid–treated group (P = .07).

Citation: American Journal of Veterinary Research 85, 6; 10.2460/ajvr.23.12.0280

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Histologic evaluation of the rat tissues collected at the termination of the study did not reveal any significant lesions within the pelvic limb musculature from the injection sites in any of the experimental groups. Two rats, 1 control animal and 1 plasmid-treated animal, had mild focal lesions of myositis or myodegeneration in the inoculation site of the caudal hindlimb musculature characterized by a mild mononuclear inflammation or focal myocyte degeneration and regeneration. These lesions in the skeletal muscle were considered to be incidental (Supplementary Figure S2). One rat in the plasmid-treatment group died idiopathically 2 days prior to euthanizing the remaining rats. The cause of death for this animal was not determined from either the subsequent gross or microscopic examinations.

EPO-Deficient anemic mice treated with Vector D lentivirus demonstrate a significant and variably persistent increase in blood PCV relative to controls

For the genetically EPO-deficient anemic mouse studies, mouse genotypes were confirmed by demonstrating that the individuals enrolled in the study were Hom for the SV40 transgene inserted into the murine EPO promoter allele (Hom EPO disrupted). The DNA from each group (eg, WT, Het, and Hom) were utilized in PCR reactions with either primers designed to amplify the WT murine EPO gene or the SV40 mutant gene. Figure 4 demonstrates mouse genotyping agarose gel electrophoresis of PCR amplicons using DNA obtained from WT mice (PCR amplicon only evident with WT primers), Het mice (PCR amplicons present with both WT and SV40 mutant primers), and Hom mutant mice (PCR amplicon only evident with the SV40 mutant primers).

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Genotyping of mice and effect of Vector D lentivirus inoculation on EPO-deficient mouse PCV. A—Agarose gel electrophoresis depicting PCR genotyping results for wild-type (WT) mice, for heterozygous mice (Het), and for mice homozygous for disrupted EPO (Hom). All of the mice have the appropriately sized PCR amplicons. A negative control (H₂O; water template) was used for both sets of primers. B—Mean blood PCV for mice treated with either the control vector or Vector D lentivirus over time. Mice treated with Vector D had significantly elevated mean PCV for weeks 1 through 5 compared to control mice (Student t test; P = .0002, .0001, .0001, .0001, and .0292, respectively).

Citation: American Journal of Veterinary Research 85, 6; 10.2460/ajvr.23.12.0280

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In the preliminary murine study (experiment #1), the inoculating dose of Vector D lentivirus was 7.8 X 10⁷ TU/mouse. PCV values were determined for 2 weeks prior to transduction. Anemic mice treated IM with Vector D lentivirus had a significantly increased mean PCV relative to control mice treated with the control vector lentivirus that persisted for 5 weeks postinoculation (mean PCV values for treated vs untreated mice ± SD [Figure 4; Table 1]). Two of the Vector D–treated mice had elevated PCV values out to 9 weeks postinoculation relative to the untreated animals in the Control group.

The second murine study was designed to determine 1) the effect of a higher inoculating dose of Vector D lentivirus (1 X 10⁸ TU/mouse), 2) the effect of a second administration of Vector D lentivirus after the PCV returned to near baseline (< 25%), and 3) the toxicity of the administration of GCV 3 weeks after initial Vector D administration. Similar to the result of the first murine study, mice treated with the escalated dose of Vector D lentivirus had a significantly elevated PCV relative to control mice for 5 weeks postinoculation (Figure 5; Table 2). However, the magnitude of the effect on PCV between the 2 studies was markedly different. For the first study, the lentivirus-treated group had a 51% mean increase in PCV (30.25%) over the control group, while in the second study, there was a 144% increase in mean PCV (47%) relative to the control group (at peak elevation in PCV, 3 weeks postinoculation).

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EPO-deficient mice administered Vector D once and then readministered Vector D after PCV returns to baseline. A—PCV over time postadministration of Vector D once. Bar with asterisks represents weeks in which there was a significant difference in PCV between the 2 groups (Student t test; weeks 2 through 6 postadministration; P < .0001, .0002, .0002, .0019, and .0087, respectively). B—PCV for the same mice after receiving a second dose of Vector D lentivirus, indicating no increase in PCV relative to control animals. The red arrow indicates a single mouse with a steadily declining PCV after readministration of Vector D and the time point of euthanasia (4 weeks post Vector D readministration). C—Representative histologic images with high-power insets of spleen and tibia with bone marrow from a control group mouse (left images) and mouse that developed red cell aplasia (right images). Scale bar, 200 μm.

Citation: American Journal of Veterinary Research 85, 6; 10.2460/ajvr.23.12.0280

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Interestingly, for mice reinoculated with the same dose of Vector D lentivirus (Group B), serial weekly PCV assessments failed to identify any evidence that the second dose of Vector D resulted in an increase in PCV relative to control animals (Figure 5). A single reinoculated mouse exhibited lethargy, pale mucous membranes, and progressive weight loss. PCV analyses demonstrated a marked and progressive anemia for this animal. The mouse was euthanized 4 weeks postreinoculation (4% PCV nadir at the time of euthanasia). Histologic evaluation of the mouse’s tissues identified an increase in brown intracytoplasmic pigment within macrophages of the spleen, consistent with blood-derived hemosiderin deposition (Figure 5). The bone marrow had a marked decrease in hematopoietic precursor cellularity, consistent with a nonregenerative anemia. The few hematopoietic cells that were identified were most consistent with myeloid lineage cells rather than erythroid precursors (Figure 5). These clinical and pathological findings are consistent with the development of red cell aplasia and associated splenic sequestration of hemosiderin.

The third objective of murine experiment #2, to test the in vivo toxicity of IP GCV, was assessed 3 weeks after administration of Vector D lentivirus. At 3 weeks postinoculation, while the mean PCV was still elevated relative to control mice (47.4% vs 20%), this murine cohort was treated IP for 5 consecutive days with 50 mg GCV/kg body weight. No evidence of weight loss or behavioral changes were identified in GCV-treated mice relative to controls, consistent with a lack of overt toxicity. A precipitous decline in PCV was not identified in the GCV-treated mice, suggesting that the administered dose of GCV did not abrogate the effect of Vector D lentivirus. However, 2 of the Vector D–inoculated and subsequent GCV-treated mice had a markedly prolonged elevation in PCV values relative to the non–GCV-treated mice (Group B), with 1 GCV-treated mouse maintaining an elevation in PCV for approximately 4.5 months post-treatment and the other approximately 7.5 months post-treatment (Figure 6).

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Mice receiving Vector D at the week 0 time point and PCV over time. A—Red arrow indicates time point at which one group (+GCV) received ganciclovir for 5 consecutive days IP at 50 mg/kg. The gray horizontal bar represents the approximate average PCV for control mice (approx 20%). B—Histologic images with higher-power images (insets) of the testis from a control mouse (left) and severe tubular degeneration and atrophy in an experimental mouse that received GCV treatment. Scale bar, 200 μm.

Citation: American Journal of Veterinary Research 85, 6; 10.2460/ajvr.23.12.0280

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Histologic evaluation of tissues collected from male mice treated with GCV demonstrated bilateral, severe tubular degeneration and atrophy within the seminiferous tubules of the testes. These lesions were characterized by loss of germ cells (spermatogonia) with preservation of supportive Sertoli cells (Figure 6). No gross or microscopic lesions were identified within the female mice receiving GCV, and no other relevant or significant histologic findings were identified in any of the mouse groups. GCV has been reported to cause tubular degeneration and atrophy of spermatogonia.⁴³ The histological findings of testicular tubular degeneration confirm that the GCV was administered at a pharmacologically appropriate dose and was associated with tissue-specific cytotoxicity but was not associated with systemic toxicity.