Anterior Cruciate Ligament Reconstruction Augmentation With Bone Marrow Aspirate Concentrate, Demineralized Bone Matrix, and Suture Tape Shows No Difference in Outcomes-But Faster Functional Recovery-Versus Non-augmented Anterior Cruciate Ligament Reconstruction.

PURPOSE: To compare outcomes after anterior cruciate ligament reconstruction (ACLR) with bone marrow aspirate concentrate (BMAC), demineralized bone matrix (DBM), and suture tape augmentation (STA) versus ACLR without biological augmentation or STA. METHODS: We performed a prospective randomized controlled trial at a single institution to compare ACLR with BMAC, DBM, and STA (group A) versus ACLR without biological augmentation or STA (group NA). The study sought to include 100 patients. Skeletally mature patients younger than 25 years received quadriceps tendon autograft, whereas patients aged 25 years or older underwent allograft ACLR with an all-inside technique. Patients with concomitant meniscal pathologies were included. The primary outcomes compared were range of motion (ROM), limb symmetry, and patient-reported outcomes. Secondary outcomes included radiographic outcomes and surgical complications. Univariate and mixed-model regression analyses were used to compare outcomes. RESULTS: Fifty-nine patients were included (29 patients in group A [11 female patients, 38%] and 30 patients in group NA [15 female patients, 50%]). Early ROM at 6 weeks (125° of flexion vs 109° of flexion, P < .0001) and limb symmetry at 12 weeks (80.6% vs 36.7% [delta, 43.9%], P < .001) were significantly improved in group A. At 2 years, International Knee Documentation Committee scores were similar (91.1 ± 12.7 vs 85.3 ± 10.8, P = .109). Quality-of-life subscores of the Knee Injury and Osteoarthritis Outcome Score were significantly enhanced in group A (85.2 ± 20.9 vs 72.1 ± 20.4, P = .042). In 22 patients (12 in group A and 10 in group NA), computed tomography scans were obtained at 6 months to compare bone tunnel healing. Overall, the mean increase in bone tunnel diameter was significantly smaller in group A than in group NA. No difference in graft rerupture or reoperation rate was observed. Reoperations were performed for stiffness in 7 of 59 patients (11.9%) (3 [10%] in group A vs 4 [13%] in group NA; P > .999). CONCLUSIONS: There were no differences in International Knee Documentation Committee scores between groups at 2-year follow-up. Functional outcomes including early ROM and limb symmetry were significantly improved in patients who received ACLR with BMAC, DBM, and STA. LEVEL OF EVIDENCE: Level II, randomized controlled trial.

A Three-dimensional Comparison of Pre- and Post-component Position in a Series of Off-label Robotic-assisted Revision Total Knee Arthroplasties.

Background: The application of robotic-assisted arthroplasty in revision knee scenarios continues to evolve. This study compares the pre- and post-revision implant positions in series of revision total knee arthroplasties (TKA) using a robotic arm system. Methods: Twenty-five consecutive off-label robotic-assisted revision TKA were performed. After virtual revision femoral and tibial components were positioned to achieve "balanced" medial and lateral flexion and extension gaps, the existing primary implants (PI) were removed, and bone cuts were executed with the robotic arm system. Preoperative coronal, sagittal, and axial position of the PI was compared to the final planned positions of the robotic revision implants (RRI) for each subject. A repeated measures ANOVA using the absolute difference in millimeters and degrees between the PI and RRI orientation was completed. Results: Intra-operatively, the virtual gaps were balanced within the planning software followed by successful execution of the plan. There was a statistically significant difference between posterior condylar offset and tibial component positioning for RRI compared to PI. There was no difference between the distal femoral component values between PI and RRI. Conclusions: The sagittal alignment of the revision implants, specifically the femoral posterior condylar offset and tibial component slope, are statistically significant considerations for a stable revision TKA with off-label use of a robotic-arm system. Other potential benefits may include appropriate implant sizing which can affect the resultant ligamentous tension important for a functional revision TKA. Future research and software iterations will be needed to determine the overall accuracy and utility of robotic-assisted revision TKA.

An Update on Physical Therapy Adjuncts in Orthopedics.

Physical therapy is a necessary part of the recovery process after most orthopedic procedures. Effective treatment, patient satisfaction, and financial reimbursement hinge on the successful implementation of both surgical and nonsurgical interventions. Evidence-based practice and open communication between therapists and orthopedic surgeons continue to form the foundation of patient care. The aim of this paper is to familiarize orthopedic surgeons with the relevant data behind some of the recent advances in rehabilitation adjuncts to better address the needs of our patients. Although each intervention has been found to be relatively safe, high-quality evidence is still sparse. Opportunities exist for improved outcomes with further well-designed studies to investigate the role of these therapy modalities among orthopedic patients.

Rotator Cuff Repair Using a Needle Arthroscope Through a Dual-Lumen Flexible Cannula.

The advent of arthroscopy in shoulder surgery has allowed for the development of minimally invasive techniques for the treatment of shoulder pathology. Further developments in needle arthroscopy have continued this trend toward less invasive shoulder surgery, allowing for decreased pain using smaller portals and decreased fluid irrigation through the shoulder joint during surgery. This technique describes a minimally invasive rotator cuff repair using a dual-lumen cannula that provides both direct visualization and direct instrument access to the pathology. This new cannula has the potential to further refine and to simplify needle arthroscopic techniques about the shoulder. With judicious patient selection, needle arthroscopy is a viable option for the treatment of common shoulder pathology.

Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: implications for the therapeutic development of RNAi.

Huntington's disease (HD) is a fatal, dominant neurodegenerative disease caused by a polyglutamine repeat expansion in exon 1 of the HD gene, which encodes the huntingtin protein. We and others have shown that RNAi is a candidate therapy for HD because expression of inhibitory RNAs targeting mutant human HD transgenes improved neuropathology and behavioral deficits in HD mouse models. Here, we developed shRNAs targeting conserved sequences in human HD and mouse HD homolog (HDh) mRNAs to initiate preclinical testing in a knockin mouse model of HD. We screened 35 shRNAs in vitro and subsequently narrowed our focus to three candidates for in vivo testing. Unexpectedly, two active shRNAs induced significant neurotoxicity in mouse striatum, although HDh mRNA expression was reduced to similar levels by all three. Additionally, a control shRNA containing mismatches also induced toxicity, although it did not reduce HDh mRNA expression. Interestingly, the toxic shRNAs generated higher antisense RNA levels, compared with the nontoxic shRNA. These results demonstrate that the robust levels of antisense RNAs emerging from shRNA expression systems can be problematic in the mouse brain. Importantly, when sequences that were toxic in the context of shRNAs were placed into artificial microRNA (miRNA) expression systems, molecular and neuropathological readouts of neurotoxicity were significantly attenuated without compromising mouse HDh silencing efficacy. Thus, miRNA-based approaches may provide more appropriate biological tools for expressing inhibitory RNAs in the brain, the implications of which are crucial to the development of RNAi for both basic biological and therapeutic applications.

Use of flow cytometry for characterization of human cytomegalovirus vaccine particles.

Despite a 40-year effort, an effective vaccine against human cytomegalovirus (HCMV) remains an unmet medical need. The discovery of potent neutralizing epitopes on the pentameric gH complex (gH/gL/UL128/130/131) has reenergized HCMV vaccine development. Our whole-virus vaccine candidate, currently in a Phase I clinical trial, is based on the attenuated AD169 strain with restored expression of the pentameric gH complex. Given the complexity of a whole-virus vaccine, improved analytical methods have been developed to better characterize heterogeneous viral particles released from infected cells during vaccine production. Here we show the utility of a commercial flow cytometer for the detection of individual HCMV particles, either via light scattering or using fluorescence after labeling of specific antigens. Rapid measurements requiring minimal material provide near real-time information on particle concentration, distributions of different particle types, and product purity. Additionally, utilizing immunoreagents has allowed us to characterize the distribution of key antigens across individual particles and particle types.

Production by Bacillus pumilus (MSH) of an antifungal compound that is active against Mucoraceae and Aspergillus species: preliminary report.

A compound produced by Bacillus pumilus (MSH) that inhibits Mucoraceae and Aspergillus species is described. Fungicidal activity was demonstrated by lawn-spotting and by diffusion through 0.45 microm Millipore membranes placed on 5 % sheep-blood agar, nutrient agar, trypticase soy agar and Mueller-Hinton agar, followed by spore inoculation of the bacterium-free underlying agar surface. With either technique, zones of fungal inhibition correlated with the zone of haemolysis produced by B. pumilus (MSH). The active compound inhibited Mucor and Aspergillus spore germination and aborted elongating hyphae, presumably by inducing a cell-wall lesion. Antifungal activity was stable in agar for a minimum of 8 days, resistant to Pronase degradation, and partially inactivated by chloroform exposure and at pH 5.6. Its molecular mass was determined by diffusion through dialysis membrane to be 500-3000 Da. Attempts at further isolation of the compound have proven unsuccessful to date.

Manufacturing recombinant adeno-associated viral vectors from producer cell clones.

A commercial rAAV manufacturing process needs to provide a safe product at high yield, be easily scalable, regulatory-compliant, and have reasonable cost of goods. Considerations for process development include not only product quantity and quality, but also ease of obtaining equipment, performing validation, and demonstrating control. In these regards, it is usually efficient to make use of proven technologies for more established areas of manufacturing, such as cell culture and purification methods used by the recombinant protein/monoclonal antibody industry. We have focused on stable mammalian producer cell lines with adenovirus type 5 helper virus as a means of achieving these goals. This review describes our current approach for generating producer cell clones and designing a scalable, regulatory-compliant vector production and purification process that addresses any product safety concerns relating to helper virus. To date, a producer cell line-based manufacturing process has been implemented at the 250-liter production scale, with no foreseeable impediments to scaling up to commercial vector manufacturing in 2000-liter bioreactors or larger.

Characterizing clearance of helper adenovirus by a clinical rAAV1 manufacturing process.

Recombinant adeno-associated viral vectors (rAAV) are being developed as gene therapy delivery vehicles and as genetic vaccines, and some of the most scaleable manufacturing methods for rAAV use live adenovirus to induce production. One aspect of establishing safety of rAAV products is therefore demonstrating adequate and reliable clearance of this helper virus by the vector purification process. The ICH Q5A regulatory guidance on viral safety provides recommendations for process design and characterization of viral clearance for recombinant proteins, and these principles were adapted to a rAAV serotype 1 purification process for clinical vectors. Specific objectives were to achieve overall adenovirus clearance factors significantly greater than input levels by using orthogonal separation and inactivation methods, and to segregate adenovirus from downstream operations by positioning a robust clearance step early in the process. Analytical tools for process development and characterization addressed problematic in-process samples, and a viral clearance validation study was performed using adenovirus and two non-specific model viruses. Overall clearance factors determined were >23 LRV for adenovirus, 11 LRV for BVDV, and >23 LRV for AMuLV.

Reversal of cardiac dysfunction after long-term expression of SERCA2a by gene transfer in a pre-clinical model of heart failure.

OBJECTIVES: The aim of this study was to examine the effects of sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) gene transfer in a swine heart failure (HF) model. BACKGROUND: Reduced expression and activity of SERCA2a have been documented in HF. Prior studies have reported the beneficial effects of short-term SERCA2a overexpression in rodent models. However, the effects of long-term expression of SERCA2a in pre-clinical large animal models are not known. METHODS: Yorkshire-Landrace pigs were used (n = 16) to create volume overload by percutaneously severing chordae tendinae of the mitral apparatus with a bioptome to induce mitral regurgitation. At 2 months, pigs underwent intracoronary delivery of either recombinant adeno-associated virus type 1 (rAAV1) carrying SERCA2a under a cytomegalovirus promoter (rAAV1.SERCA2a) (n = 10; group 1) or saline (n = 6; group 2). RESULTS: At 2 months, study animals were found to be in a compensated state of volume-overload HF (increased left ventricular internal diastolic and systolic diameters [LVIDd and LVIDs]). At 4 months, gene transfer resulted in: 1) positive left ventricular (LV) inotropic effects (adjusted peak left ventricular pressure rate of rise (dP/dt)max/P, 21.2 +/- 3.2 s(-1) group 1 vs. 15.5 +/- 3.0 s(-1) group 2; p < 0.01); 2) improvement in LV remodeling (% change in LVIDs -3.0 +/- 10% vs. +15 +/- 11%, respectively; p < 0.01). At follow-up, brain natriuretic peptide levels remained stable in group 1 after gene transfer, in contrast to rising levels in group 2. Further, cardiac SERCA2a expression was significantly decreased in group 2 whereas in group 1 it was restored to normal levels. There was no histopathological evidence of acute myocardial inflammation or necrosis. CONCLUSIONS: Using a large-animal, volume-overload model of HF, we report that long-term overexpression of SERCA2a by in vivo rAAV1-mediated intracoronary gene transfer preserved systolic function, potentially prevented diastolic dysfunction, and improved ventricular remodeling.

Restoration of mechanical and energetic function in failing aortic-banded rat hearts by gene transfer of calcium cycling proteins.

The aim of this study was to examine whether short- and long-term gene transfer of Ca(2+) handling proteins restore left ventricular (LV) mechanoenergetics in aortic banding-induced failing hearts. Aortic-banded rats received recombinant adenoviruses carrying sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) (Banding+SERCA), parvalbumin (Banding+Parv) or beta-galactosidase (Banding+betagal), or an adeno-associated virus carrying SERCA2a (Banding+AAV.SERCA) by a catheter-based technique. LV mechanoenergetic function was measured in cross-circulated hearts. "Banding", "Banding+betagal" and "Banding+saline" groups showed lower end-systolic pressure at 0.1 ml intraballoon water (ESP(0.1)), higher end-diastolic pressure at 0.1 ml intraballoon water (EDP(0.1)) and slower LV relaxation rate, compared with "Normal" and "Sham". However, "Banding+SERCA" and "Banding+Parv" showed high ESP(0.1), low EDP(0.1) and fast LV relaxation rate. In "Banding", "Banding+betagal" and "Banding+saline", slope of relation between cardiac oxygen consumption and systolic pressure-volume area, O(2) cost of total mechanical energy, was twice higher than normal value, whereas slope in "Baning+SERCA" and "Banding+Parv" was similar to normal value. Furthermore, O(2) cost of LV contractility in the 3 control banding groups was approximately 3 times higher than normal value, whereas O(2) cost of contractility in "Banding+SERCA", "Banding+AAV.SERCA" and "Banding+Parv" was as low as normal value. Thus, high O(2) costs of total mechanical energy and of LV contractility in failing hearts indicate energy wasting both in chemomechanical energy transduction and in calcium handling. Improved calcium handling by both short- and long-term overexpression of SERCA2a and parvalbumin transforms the inefficient energy utilization into a more efficient state. Therefore enhancement of calcium handling either by resequestration into the SR or by intracellular buffering improves not only mechanical but energetic function in failing hearts.

Production of recombinant adeno-associated type 5 (rAAV5) vectors using recombinant herpes simplex viruses containing rep and cap.

We developed a scaleable production system for adeno-associated virus type 5 (AAV5)-based vectors using a replication-defective recombinant herpes simplex type 1 virus (rHSV) containing the rep and cap genes of AAV5. Multiple rHSV isolates containing AAV5 rep and cap with normal or altered p5 promoter elements were constructed and tested in vector production. Compared with rAAV5 vector yields obtained by plasmid transfection, yields of rAAV5 using any of the rHSV isolates were low. Evidence suggests that the low vector yields are a consequence of the extensive and early cytopathology induced by the rHSV isolates. In addition, we found a correlation between the amount of Rep52 or Rep40 proteins and the amount of vector produced by each rHSV isolate, suggesting that packaging of vector DNA into virus particles is rate-limiting when using rHSV to generate rAAV5 vectors.

Secretion of a TNFR:Fc fusion protein following pulmonary administration of pseudotyped adeno-associated virus vectors.

This study evaluated and compared delivery of the tumor necrosis factor alpha receptor (TNFR)-immunoglobulin G1 (IgG1) Fc fusion (TNFR:Fc) gene to the lung by single and repeat administrations of multiple pseudotyped adeno-associated virus (AAV) vectors as a means for achieving systemic distribution of the soluble TNFR:Fc protein. A single endotracheal administration of AAV[2/5]cytomegalovirus (CMV)-TNFR:Fc vector (containing the AAV2 inverted terminal repeats and AAV5 capsid) to the rat lung resulted in long-term, high levels of serum TNFR:Fc protein that gradually declined over a period of 8 months. Endotracheal delivery of AAV[2/1]CMV-TNFR:Fc resulted in serum TNFR:Fc protein levels that were detectable for at least 4 months but were 10-fold lower than that of the AAV[2/5] vector. In contrast, secretion of the TNFR:Fc protein following pulmonary delivery of AAV[2/2]CMV-TNFR:Fc vector was very inefficient, and the protein was detected in the blood only when an airway epithelial cell-specific promoter, CC10, was substituted for the CMV enhancer/promoter to control transgene expression. In the context of AAV[2/5], the CC10 promoter was as efficient as CMV enhancer/promoter in generating similar levels of systemic TNFR:Fc protein, suggesting that this protein is secreted primarily from the airway epithelium. In mice, comparable long-term secretion of TNFR:Fc protein was demonstrated after AAV[2/2] and AAV[2/5] delivery, although the kinetics of transduction appeared to be different. All pseudotyped AAV vectors elicited serum anti-AAV capsid-neutralizing antibody responses, but these did not prevent lung transduction and efficient secretion of TNFR:Fc protein to the circulation following readministration with AAV[2/5]. These results highlight the potential utility of AAV vectors containing serotype 5 capsid to deliver and redeliver genes of secreted proteins to the lung to achieve long-term systemic protein expression.

Distinct classes of proteasome-modulating agents cooperatively augment recombinant adeno-associated virus type 2 and type 5-mediated transduction from the apical surfaces of human airway epithelia.

Tripeptidyl aldehyde proteasome inhibitors have been shown to effectively increase viral capsid ubiquitination and transduction of recombinant adeno-associated virus type 2 (rAAV-2) and rAAV-5 serotypes. In the present study we have characterized a second class of proteasome-modulating agents (anthracycline derivatives) for their ability to induce rAAV transduction. The anthracycline derivatives doxorubicin and aclarubicin were chosen for analysis because they have been shown to interact with the proteasome through a mechanism distinct from that of tripeptidyl aldehydes. Our studies demonstrated that doxorubicin and aclarubicin also significantly augmented rAAV transduction in airway cell lines, polarized human airway epithelia, and mouse lungs. Both tripeptidyl aldehyde and anthracycline proteasome-modulating agents similarly augmented nuclear accumulation of rAAV in A549 and IB3 airway cell lines. However, these two cell types demonstrated cell specificity in the ability of N-acetyl-L-leucyl-L-leucyl-L-norleucine (LLnL) or doxorubicin to augment rAAV transduction. Interestingly, the combined administration of LLnL and doxorubicin resulted in substantially increased transduction (>2,000-fold) following apical infection of human polarized epithelia with either rAAV-2 or rAAV-5. In summary, the cell type specificity of LLnL and doxorubicin to induce rAAV transduction, together with the ability of these compounds to synergistically enhance rAAV transduction in polarized airway epithelial induction, suggests that these two classes of compounds likely modulate different proteasome functions that affect rAAV transduction. Findings from this study provide new insights into how modulation of proteasome function can be effectively used to augment rAAV transduction in airway epithelia for gene therapy of cystic fibrosis.

Dual therapeutic utility of proteasome modulating agents for pharmaco-gene therapy of the cystic fibrosis airway.

Pharmacologic- and gene-based therapies have historically been developed as two independent therapeutic platforms for cystic fibrosis (CF) lung disease. Inhibition of the dysregulated epithelial Na channel (ENaC) is one pharmacologic approach to enhance airway clearance in CF. We investigated pharmacologic approaches to enhance CFTR gene delivery with recombinant adeno-associated virus (rAAV) and identified compounds that significantly improved viral transduction while simultaneously inhibiting ENaC activity through an unrelated mechanism. Treatment of human CF airway epithelia with proteasome modulating agents (LLnL and doxorubicin) at the time of rAAV2 or rAAV2/5 infection dramatically enhanced CFTR gene delivery and correction of CFTR-mediated short-circuit currents. Surprisingly, these agents also facilitated long-term (15-day) functional inhibition of ENaC currents independent of CFTR vector administration. Inhibition of ENaC activity was predominantly attributed to a doxorubicin-dependent decrease in gamma-ENaC subunit mRNA expression and an increase in gamma-ENaC promoter methylation. This is the first report to describe the identification of compounds with dual therapeutic action that are able to enhance the efficacy of CFTR gene therapy to the airway while simultaneously ameliorating primary aspects of CF disease pathophysiology. The identification of such compounds mark a new area for drug development, not only for CF, but also for other gene therapy disease targets.

AAV2 vector harboring a liver-restricted promoter facilitates sustained expression of therapeutic levels of alpha-galactosidase A and the induction of immune tolerance in Fabry mice.

The successful application of gene therapy for the treatment of genetic diseases such as Fabry is reliant on the development of vectors that are safe and that facilitate sustained expression of therapeutic levels of the transgene product. Here, we report that intravenous administration of a recombinant AAV2 vector encoding human alpha-galactosidase A under the transcriptional control of a liver-restricted enhancer/promoter (AAV2/DC190-alphagal) generated significantly higher levels of expression in BALB/c and Fabry mice than could be realized using the ubiquitous CMV promoter (AAV2/CMVHI-alphagal). Moreover, AAV2/DC190-alphagal-mediated hepatic expression of alpha-galactosidase A was sustained for 12 months in BALB/c mice and was associated with a significantly reduced immune response to the expressed enzyme. Subsequent challenge of the AAV2/DC190-alphagal-treated animals with recombinant human alpha-galactosidase A at 6 months failed to elicit the production of anti-alpha-galactosidase A antibodies, suggesting the induction of immune tolerance in these animals. The levels of expression attained with AAV2/DC190-alphagal in the Fabry mice were sufficient to reduce the abnormal accumulation of globotriaosylceramide in the liver, spleen, and heart to basal levels and in the kidney by approximately 40% at 8 weeks. Together, these results demonstrate that AAV2-mediated gene transfer that limits the expression of alpha-galactosidase A to the liver may be a viable strategy for treating Fabry disease.