Efficacy of Liposomal Bupivacaine Infiltration on the Management of Total Knee Arthroplasty.

Importance: Liposomal bupivacaine is a novel extended-duration anesthetic that has recently been used for local infiltration in total knee arthroplasty (TKA). Athough liposomal bupivacaine is widely used, it is unknown if the benefits justify the cost in the veteran population at our institution. Objective: To evaluate a change in practice: the effect of local infiltration of liposomal bupivacaine on perioperative outcomes in patients undergoing primary TKA. Design, Setting, and Participants: A retrospective cohort study was conducted among patients who underwent primary TKA at a Veterans Affairs Medical Center before (March 3, 2013-March 2, 2014) and after (March 3, 2014-March 2, 2015) the implementation of liposomal bupivacaine for local infiltration in TKA. Intervention: Drug utilization evaluation of liposomal bupivacaine for local infiltration in TKA. Main Outcomes and Measures: Use of opioids after discharge from the postanesthesia care unit. Results: Among 199 patients, those who received liposomal bupivacaine after primary TKA (mean [SD] age, 65.3 [6.9] years; 93 males and 5 females) had a reduced median opioid use in the first 24 hours after surgery compared with those who did not receive liposomal bupivacaine (mean [SD] age, 64.9 [8.4] years; 95 males and 6 females; [intravenous morphine equivalents, 12.50 vs 22.50 mg; P = .001]). The use of patient-controlled analgesia was also reduced among patients who received liposomal bupivacaine vs those who did not (49 vs 91; P < .001). A reduction in the use of antiemetics was observed in the first 24 hours after surgery (13 vs 34; P = .001) and in the postanesthesia care unit among those who received liposomal bupivacaine vs those who did not (4 vs 20; P = .001). The number of patients in the postanesthesia care unit with no pain was improved among those who received liposomal bupivacaine vs those who did not (44 vs 19; P < .001). Although median (interquartile range) pain scores in the postanesthesia care unit were improved among patients who received liposomal bupivacaine vs those who did not (4.0 [0.0-6.6] vs 5.5 [3.0-7.5]; P = .001), patients who received liposomal bupivacaine had greater median (interquartile range) pain scores 48 hours (5.5 [4.0-7.0] vs 5.0 [3.0-6.0]; P = .01), 72 hours (5.0 [4.0-6.0] vs 4.0 [2.0-6.0]; P = .002), and 96 hours (5.0 [3.0-6.5] vs 4.0 [1.0-5.0]; P = .003) after surgery than those who did not receive liposomal bupivacaine. There was no difference in the median length of stay between the 2 groups. Institutional cost savings was estimated at $27 000 per year. Conclusions and Relevance: Local infiltration of liposomal bupivacaine reduces use of opioids in the first 24 hours after primary TKA. Similarly, reduction in antiemetic use and improved postoperative pain are also seen in the first 24 hours after surgery but are limited to this time frame. Furthermore, a positive institutional cost savings was observed.

Long-term follow-up after gene therapy for canavan disease.

Canavan disease is a hereditary leukodystrophy caused by mutations in the aspartoacylase gene (ASPA), leading to loss of enzyme activity and increased concentrations of the substrate N-acetyl-aspartate (NAA) in the brain. Accumulation of NAA results in spongiform degeneration of white matter and severe impairment of psychomotor development. The goal of this prospective cohort study was to assess long-term safety and preliminary efficacy measures after gene therapy with an adeno-associated viral vector carrying the ASPA gene (AAV2-ASPA). Using noninvasive magnetic resonance imaging and standardized clinical rating scales, we observed Canavan disease in 28 patients, with a subset of 13 patients being treated with AAV2-ASPA. Each patient received 9 × 10(11) vector genomes via intraparenchymal delivery at six brain infusion sites. Safety data collected over a minimum 5-year follow-up period showed a lack of long-term adverse events related to the AAV2 vector. Posttreatment effects were analyzed using a generalized linear mixed model, which showed changes in predefined surrogate markers of disease progression and clinical assessment subscores. AAV2-ASPA gene therapy resulted in a decrease in elevated NAA in the brain and slowed progression of brain atrophy, with some improvement in seizure frequency and with stabilization of overall clinical status.

Intracranial glioblastoma models in preclinical neuro-oncology: neuropathological characterization and tumor progression.

Although rodent glioblastoma (GBM) models have been used for over 30 years, the extent to which they recapitulate the characteristics encountered in human GBMs remains controversial. We studied the histopathological features of dog GBM and human xenograft GBM models in immune-deficient mice (U251 and U87 GBM in nude Balb/c), and syngeneic GBMs in immune-competent rodents (GL26 cells in C57BL/6 mice, CNS-1 cells in Lewis rats). All GBMs studied exhibited neovascularization, pleomorphism, vimentin immunoreactivity, and infiltration of T-cells and macrophages. All the tumors showed necrosis and hemorrhages, except the U87 human xenograft, in which the most salient feature was its profuse neovascularization. The tumors differed in the expression of astrocytic intermediate filaments: human and dog GBMs, as well as U251 xenografts expressed glial fibrillary acidic protein (GFAP) and vimentin, while the U87 xenograft and the syngeneic rodent GBMs were GFAP(-) and vimentin(+). Also, only dog GBMs exhibited endothelial proliferation, a key feature that was absent in the murine models. In all spontaneous and implanted GBMs we found histopathological features compatible with tumor invasion into the non-neoplastic brain parenchyma. Our data indicate that murine models of GBM appear to recapitulate several of the human GBM histopathological features and, considering their reproducibility and availability, they constitute a valuable in vivo system for preclinical studies. Importantly, our results indicate that dog GBM emerges as an attractive animal model for testing novel therapies in a spontaneous tumor in the context of a larger brain.

Efficacy of nonviral gene transfer in the canine brain.

OBJECT: The purpose of this study was to evaluate the gene transfer capability and tolerability of plasmid DNA/polyethylenimine (PEI) complexes in comparison with adenovirus and naked plasmid DNA in the canine brain. METHODS: Plasmid or adenoviral vectors encoding firefly luciferase were injected directly into the cerebral parenchyma of five adult dogs at varying doses and volumes. Serial physical and neurological examinations, as well as blood and cerebrospinal fluid (CSF) analyses, were conducted before and after the surgery for 3 days. Three days after gene delivery, a luciferase activity assay and immunofluorescence analysis were used to test the brain tissue for gene expression. RESULTS: Injection into the brain parenchyma resulted in gene transfer throughout the cerebrum with every vector tested. Luciferase expression was highest when adenovirus vectors were used. Injection of plasmid DNA/PEI complexes and naked DNA resulted in similar levels of luciferase expression, which were on average 0.5 to 1.5% of the expression achieved with adenovirus vectors. Immunofluorescent microscopy analysis revealed that plasmid DNA/PEI complexes transduced mainly neurons, whereas adenovirus transduced mainly astrocytes. No significant acute side effects or neurological complications were observed in any of the dogs. Mononuclear cell counts significantly increased in the CSF after adenovirus injection and modestly increased after injection of plasmid DNA/PEI complexes, suggesting that a mild, acute inflammatory response occurred in the central nervous system (CNS). CONCLUSIONS: Compared with rodent models that are limited by very small brains, the dog is an excellent preclinical model in which to assess the distribution and safety of emerging gene transfer technologies. In this study, short-term gene transfer was evaluated as a prelude to long-term expression and safety studies. The authors conclude that the viral and nonviral vectors tested were well tolerated and effective at mediating gene transfer throughout a large portion of the canine brain. The nonviral plasmid vectors were less effective than adenovirus, yet they still achieved appreciable gene expression levels. Due to reduced gene transfer efficiency relative to viral vectors, nonviral vectors may be most useful when the expressed protein is secreted or exerts a bystander effect. Nonviral vectors offer an alternative means to genetically modify cells within the CNS of large mammals.

Optimization of adenoviral vector-mediated transgene expression in the canine brain in vivo, and in canine glioma cells in vitro.

Expression of the immune-stimulatory molecule Fms-like tyrosine kinase 3 ligand (Flt3L) and the conditional cytotoxic enzyme herpes simplex virus type 1 thymidine kinase (HSV1-TK) provides long-term immune-mediated survival of large glioblastoma multiforme (GBM) models in rodents. A limitation for predictive testing of novel antiglioma therapies has been the lack of a glioma model in a large animal. Dogs bearing spontaneous GBM may constitute an attractive large-animal model for GBM, which so far has remained underappreciated. In preparation for a clinical trial in dogs bearing spontaneous GBMs, we tested and optimized adenovirus-mediated transgene expression with negligible toxicity in the dog brain in vivo and in canine J3T glioma cells. Expression of the marker gene beta-galactosidase (beta-Gal) was higher when driven by the murine (m) than the human (h) cytomegalovirus (CMV) promoter in the dog brain in vivo, without enhanced inflammation. In the canine brain, beta-Gal was expressed mostly in astrocytes. beta-Gal activity in J3T cells was also higher with the mCMV than the hCMV promoter driving tetracycline-dependent (TetON) transgene expression within high-capacity adenovirus vectors (HC-Ads). Dog glioma cells were efficiently transduced by HC-Ads expressing mCMV-driven HSV1-TK, which induced 90% reduction in cell viability in the presence of ganciclovir. J3T cells were also effectively transduced with HC-Ads expressing Flt3L under the control of the regulatable TetON promoter system, and as predicted, Flt3L release was stringently inducer dependent. HC-Ads encoding therapeutic transgenes under the control of regulatory sequences driven by the mCMV promoter are excellent vectors for the treatment of spontaneous GBM in dogs, which constitute an ideal preclinical animal model.

Adenoviral-mediated gene transfer into the canine brain in vivo.

OBJECTIVE: Glioblastoma multiforme (GBM) is a devastating brain tumor for which there is no cure. Adenoviral-mediated transfer of conditional cytotoxic (herpes simplex virus [HSV] 1-derived thymidine kinase [TK]) and immunostimulatory (Fms-like tyrosine kinase 3 ligand [Flt3L]) transgenes elicited immune-mediated long-term survival in a syngeneic intracranial GBM model in rodents. However, the lack of a large GBM animal model makes it difficult to predict the outcome of therapies in humans. Dogs develop spontaneous GBM that closely resemble the human disease; therefore, they constitute an excellent large animal model. We assayed the transduction efficiency of adenoviral vectors (Ads) encoding beta-galactosidase (betaGal), TK, and Flt3L in J3T dog GBM cells in vitro and in the dog brain in vivo. METHODS: J3T cells were infected with Ads (30 plaque-forming units/cell; 72 h) encoding betaGal (Ad-betaGal), TK (Ad-TK), or Flt3L (Ad-Flt3L). We determined transgene expression by immunocytochemistry, betaGal activity, Flt3L enzyme-linked immunosorbent assay, and TK-induced cell death. Ads were also injected intracranially into the parietal cortex of healthy dogs. We determined cell-type specific transgene expression and immune cell infiltration. RESULTS: Adenoviral-mediated gene transfer of HSV1-TK, Flt3L, and betaGal was detected in dog glioma cells in vitro (45% transduction efficiency) and in the dog brain in vivo (10-mm area transduced surrounding each injection site). T cells and macrophages/activated microglia infiltrated the injection sites. Importantly, no adverse clinical or neuropathological side effects were observed. CONCLUSION: We demonstrate effective adenoviral-mediated gene transfer into the brain of dogs in vivo and support the use of these vectors to develop an efficacy trial for canine GBM as a prelude to human trials.

Mild-onset presentation of Canavan's disease associated with novel G212A point mutation in aspartoacylase gene.

We describe two sisters with a mild-onset variant of Canavan's disease who presented at age 50 and 19 months with developmental delay but without macrocephaly, hypotonia, spasticity, or seizures. Remarkably, both patients had age-appropriate head control, gross motor development, and muscle tone. There were very mild deficits in fine motor skills, coordination, and gait. Both sisters had a history of strabismus, but otherwise vision was normal. The older child showed evidence of mild cognitive and social impairment, whereas language and behavior were normal for age in the infant. Both patients were found to be compound heterozygotes for C914A (A305E) and G212A (R71H) mutations in ASPA. Like all other known ASPA mutations, this previously unknown G212A mutation appears to have low absolute enzyme activity. Nevertheless, it is associated in these patients with an extremely benign phenotype that is highly atypical of Canavan's disease. Biochemical and clinical data were evaluated using a generalized linear mixed model generated from 25 other subjects with Canavan's disease. There were statistically significant differences in brain chemistry and clinical evaluations, supporting a distinct variant of Canavan's disease. Future studies of ASPA enzyme structure and gene regulation in these subjects could lead to a better understanding of Canavan's pathophysiology and improvements in ASPA gene therapy.

Emerging concepts in glioma biology: implications for clinical protocols and rational treatment strategies.

Glioblastoma multiforme (GBM), the most common primary central nervous system neoplasm, is a complex, heterogeneous disease. The recent identification of stem cells in murine tumor xenografts that were capable of recapitulating the tumor phenotype adds a new dimension of complexity to the already challenging treatment of patients with GBMs. Although specific cellular and genetic changes are commonly associated with GBM, the mechanism by which those changes occur may have a significant impact on treatment outcome. Of the many bioinformatics techniques developed in recent years, gene expression profiling has become a commonly used research tool for investigating tumor characteristics, and the development of rationally targeted molecular therapies has also accelerated following the initial success of specifically designed inhibitors in the treatment of malignancies. Despite these advances in research techniques and targeted molecular therapies, however, limited clinical impact has been achieved in the treatment of infiltrative malignancies such as GBMs. Thus, further extension in survival of patients with GBMs may require use of multiple analyses of tumors to develop tailored therapies that reflect the inter- and intratumoral heterogeneity of this disease. In this review, the authors briefly consider the potential use of expression profiling combined with mutation analysis in the development of treatment modalities to address the heterogeneity of this complex tumor phenotype.

Combinatorial antiangiogenic gene therapy by nonviral gene transfer using the sleeping beauty transposon causes tumor regression and improves survival in mice bearing intracranial human glioblastoma.

Glioblastoma is a fatal brain tumor that becomes highly vascularized by secreting proangiogenic factors and depends on continued angiogenesis to increase in size. Consequently, a successful antiangiogenic therapy should provide long-term inhibition of tumor-induced angiogenesis, suggesting long-term gene transfer as a therapeutic strategy. In this study a soluble vascular endothelial growth factor receptor (sFlt-1) and an angiostatin-endostatin fusion gene (statin-AE) were codelivered to human glioblastoma xenografts by nonviral gene transfer using the Sleeping Beauty (SB) transposon. In subcutaneously implanted xenografts, co-injection of both transgenes showed marked anti-tumor activity as demonstrated by reduction of tumor vessel density, inhibition or abolition of glioma growth, and increase in animal survival (P = 0.003). Using luciferase-stable engrafted intracranial gliomas, the anti-tumor effect of convection-enhanced delivery of plasmid DNA into the tumor was assessed by luciferase in vivo imaging. Sustained tumor regression of intracranial gliomas was achieved only when statin-AE and sFlt-1 transposons were coadministered with SB-transposase-encoding DNA to facilitate long-term expression. We show that SB can be used to increase animal survival significantly (P = 0.008) by combinatorial antiangiogenic gene transfer in an intracranial glioma model.

Nonviral vectors for cancer gene therapy: prospects for integrating vectors and combination therapies.

Gene therapy has the potential to improve the clinical outcome of many cancers by transferring therapeutic genes into tumor cells or normal host tissue. Gene transfer into tumor cells or tumor-associated stroma is being employed to induce tumor cell death, stimulate anti-tumor immune response, inhibit angiogenesis, and control tumor cell growth. Viral vectors have been used to achieve this proof of principle in animal models and, in select cases, in human clinical trials. Nevertheless, there has been considerable interest in developing nonviral vectors for cancer gene therapy. Nonviral vectors are simpler, more amenable to large-scale manufacture, and potentially safer for clinical use. Nonviral vectors were once limited by low gene transfer efficiency and transient or steadily declining gene expression. However, recent improvements in plasmid-based vectors and delivery methods are showing promise in circumventing these obstacles. This article reviews the current status of nonviral cancer gene therapy, with an emphasis on combination strategies, long-term gene transfer using transposons and bacteriophage integrases, and future directions.

Effect of altering titer, serotype, and promoter in recombinant adenoassociate virus gene therapy expression of spinal cord neurons and astrocytes.

STUDY DESIGN: Descriptive histologic analysis of spinal cord gene therapy. OBJECTIVE: To maximize protein expression in rat spinal cord using recombinant adenoassociate virus viral vector. SUMMARY OF BACKGROUND DATA: There are few reports of spinal cord genetic transfer. There have been no reports that compare techniques to increase protein expression through genetic alterations or have illustrated successful genetic transfer to spinal cord astrocytes. METHODS: Adenoassociate virus constructs were packaged using three separate plasmids: a cis plasmid with the expression cassettes (pAM/neuron-specific enolase/green fluorescent protein/woodchuck posttranscriptional regulatory element/simian virus 40/polyadenylase or pAM/glial fibrillary acid protein/green fluorescent protein/woodchuck posttranscriptional regulatory element/simian virus 40/polyadenylase), the Ad-adenoassociate virus helper trans plasmid, and the essential region from the adenovirus genome (pFDelta6). The adenoassociate virus 2/5 capsid gene replaces the adenoassociate virus 2 capsid region in the trans construct, resulting in a different cellular tropism. Thirty-two adult (300-375 g) male Sprague-Dawley rats underwent L1 laminectomies. A total volume of 6 microL was injected directly into the spinal cord parenchyma at a rate of 600 nL/min with adenoassociate virus 2/glial fibrillary acid protein/green fluorescent protein, adenoassociate virus 2/neuron-specific enolase/green fluorescent protein, adenoassociate virus 2/5/glial fibrillary acid protein/green fluorescent protein, or adenoassociate virus 2/5/neuron-specific enolase/green fluorescent protein and either a low- (4 x 10(8)) or high-titer (1 x 10(10)) viral solution. RESULTS: The gene expression (green fluorescent protein reporter) was present in the cell bodies and axonal processes of all adenoassociate virus/green fluorescent protein constructs. However, a greater spread of virus was observed in rats injected with adenoassociate virus 2/5 compared with adenoassociate virus 2. In addition, more neurons were transduced with adenoassociate virus 2/5 than adenoassociate virus 2, and green fluorescent protein expression in neurons transduced with adenoassociate virus 2/5 appeared more intense compared with adenoassociate virus 2 neurons. The difference observed between adenoassociate virus 2 and adenoassociate virus 2/5 at 4 x 10(8) genomic particles/mL was not as profound when the virus titer was raised to 1 x 10(10) genomic particles/mL. Green fluorescent protein expression was observed in astrocytes following injection of rat spinal cords with either adenoassociate virus 2 or adenoassociate virus 2/5 carrying the glial fibrillary acid protein/green fluorescent protein construct. However, unlike neuron-specific enolase-driven expression, there was less overall expression, but a substantial increase in green fluorescent protein expression was observed with adenoassociate virus 2/5 compared with adenoassociate virus 2 with high virus titers. Furthermore, unlike the neuron-specific enolase promoter, glial fibrillary acid protein-driven expression of green fluorescent protein was not restricted to astrocytes alone. The glial fibrillary acid protein construct was able to transfect glial cells and maintain glial expression. CONCLUSION: Adenoassociate virus can readily transduce spinal cord neurons and is an efficient nonpathologic vector to deliver expression cassettes. Increased titers and the adenoassociate virus 2/5 serotype appeared to maximize expression.

Clinical protocol. Gene therapy of Canavan disease: AAV-2 vector for neurosurgical delivery of aspartoacylase gene (ASPA) to the human brain.

This clinical protocol describes virus-based gene transfer for Canavan disease, a childhood leukodystrophy. Canavan disease, also known as Van Bogaert-Bertrand disease, is a monogeneic, autosomal recessive disease in which the gene coding for the enzyme aspartoacylase (ASPA) is defective. The lack of functional enzyme leads to an increase in the central nervous system of the substrate molecule, N-acetyl-aspartate (NAA), which impairs normal myelination and results in spongiform degeneration of the brain. No effective treatment currently exists; however, virus-based gene transfer has the potential to arrest or reverse the course of this otherwise fatal condition. This procedure involves neurosurgical administration of approximately 900 billion genomic particles (approximately 10 billion infectious particles) of recombinant adeno-associated virus (AAV) containing the aspartoacylase gene (ASPA) directly to affected regions of the brain in each of 21 patients with Canavan disease. Pre- and post-delivery assessments include a battery of noninvasive biochemical, radiological, and neurological tests. This gene transfer study represents the first clinical use of AAV in the human brain and the first instance of viral gene transfer for a neurodegenerative disease.