Modulating expression of inhibitory and stimulatory immune 'checkpoints' using nanoparticulate-assisted nucleic acid delivery.

Immune checkpoints are regulatory molecules responsible for determining the magnitude and nature of the immune response. The aim of immune checkpoint targeting immunotherapy is to manipulate these interactions, engaging the immune system in treatment of cancer. Clinically, the use of monoclonal antibodies to block immunosuppressive interactions has proven itself to be a highly effective immunotherapeutic intervention. Within the literature there are numerous candidates for next generation of immune checkpoint targeting strategies. One such example is the use of nucleic acid to alter expression levels of immune checkpoint molecules, either as antisense oligo nucleotides/siRNA, to downregulate inhibitory molecules, or mRNA/DNA, to express co-stimulatory molecules. A significant component of nucleic acid delivery is its formulation within a nanoparticulate system. In this review we discuss the progress of the preclinical application of nucleic acid-based immunotherapies to target a selection of co-inhibitory/co-stimulatory molecules. Furthermore, we identify the potential and current gaps within the literature which may form the basis of future work.

Attenuated Salmonella carrying plasmid co-expressing HPV16 L1 and siRNA-E6 for cervical cancer therapy.

Human papillomavirus (HPV) infection is the major etiological factor for cervical cancer. HPV prophylactic vaccines based on L1 virus-like particles have been considered as an effective prevention method. However, existing recombination vaccines are too expensive for developing countries. DNA vaccines might be a lower-cost and effective alternative. In this study, a plasmid (pcDNA3.1-HPV16-L1) and a co-expressing plasmid (pcDNA3.1-HPV16-L1-siE6) carried by attenuated Salmonella were constructed and their prevention and treatment effect on cervical cancer were observed, respectively. The results showed that pcDNA3.1-HPV16-L1 carried by attenuated Salmonella could induce the production of HPV16-L1 antibodies, IL-2 and INF-γ in mice serum, which presented its prevention effect on HPV. Subsequently, E6 and E7 gene silencing by pCG-siE6 inhibited the growth of cervical cancer both in vitro and in vivo. Furthermore, L1 up-regulation and E6/E7 down-regulation caused by co-expressing plasmid (pcDNA3.1-HPV16-L1-siE6) contributed to a significant anti-tumor effect on the mice. This study suggests that pcDNA3.1-HPV16-L1-siE6 carried by attenuated Salmonella has a synergistic effect of immune regulation and RNA interference in cervical cancer treatment.

Exosome-mediated delivery of CRISPR/Cas9 for targeting of oncogenic KrasG12D in pancreatic cancer.

CRISPR/Cas9 is a promising technology for gene editing. To date, intracellular delivery vehicles for CRISPR/Cas9 are limited by issues of immunogenicity, restricted packaging capacity, and low tolerance. Here, we report an alternative, nonviral delivery system for CRISPR/Cas9 based on engineered exosomes. We show that non-autologous exosomes can encapsulate CRISPR/Cas9 plasmid DNA via commonly available transfection reagents and can be delivered to recipient cancer cells to induce targeted gene deletion. As a proof-of-principle, we demonstrate that exosomes loaded with CRISPR/Cas9 can target the mutant Kras G12D oncogenic allele in pancreatic cancer cells to suppress proliferation and inhibit tumor growth in syngeneic subcutaneous and orthotopic models of pancreatic cancer. Exosomes may thus be a promising delivery platform for CRISPR/Cas9 gene editing for targeted therapies.

Preparation of FA-targeted magnetic nanocomposites co-loading TFPI-2 plasmid and cis-platinum and its targeted therapy effects on nasopharyngeal carcinoma.

The majority of patients diagnosed with nasopharyngeal carcinoma (NPC) present with advanced-stage disease. The main treatment for these patients is concurrent chemoradiotherapy, which has various side effects. To improve the therapeutic effects and reduce the side effects of NPC chemoradiotherapy, we constructed a multifunctional folic acid (FA)-targeted magnetic nanocomposite codelivering tissue factor pathway inhibitor-2 (TFPI-2) and cisplatin (CDDP). This novel nanocomposite (FA-MNP/CDDP/TFPI-2) was obtained by amidation and electrostatic adsorption between FA-methoxypolyethylene glycol-polyethyleneimine (FA-MPEG-PEI) containing the TFPI-2 plasmid and magnetic nanoparticles modified by aldehyde sodium alginate loaded with CDDP. Transmission electron microscopy (TEM) images showed that the size of the individual magnetite particle core was approximately 11.5 nm. The structure and composition of the nanocomposites were identified and examined by 1H nuclear magnetic resonance (NMR) spectroscopy and ultraviolet (UV) spectrophotometry. The fluorescence analysis, Prussian blue iron staining, magnetic resonance (MR) imaging and whole-body fluorescence imaging results demonstrated that FA-MNP/CDDP/TFPI-2 showed high gene transfection efficiency and could target tumor cells via folate receptor (FR)-mediated delivery. The codelivery analysis showed that the obtained FA-MNP/CDDP/TFPI-2 composite could cause significantly more apoptosis than treatment with CDDP or TFPI-2 alone. The results showed that the FA-MNP/CDDP/TFPI-2 composites were successfully synthesized and indicated to be a specific molecular target for the FR with significant inhibitory effects on the growth of HNE-1 cells.

Tinagl1 Gene Therapy Suppresses Growth and Remodels the Microenvironment of Triple Negative Breast Cancer.

Triple negative breast cancer (TNBC) remains one of the most challenging subtypes of breast cancer to treat and is responsible for approximately 12% of breast cancer cases in the US per year. In 2019, the protein Tinagl1 was identified as a key factor for improved prognoses in certain TNBC patients. While the intracellular mechanism of action has been thoroughly studied, little is known about the role of Tinagl1 in the tumor microenvironment. In this study, we developed a lipid nanoparticle-based gene therapy to directly target the expression of Tinagl1 in tumor cells for localized expression. Additionally, we sought to characterize the changes to the tumor microenvironment induced by Tinagl1 treatment, with the goal of informing future choices for combination therapies including Tinagl1. We found that Tinagl1 gene therapy was able to slow tumor growth from the first dose and that the effects held steady for nearly a week following the final dose. No toxicity was found with this treatment. Additionally, the use of Tinagl1 increases the tumor vasculature by 3-fold but does not increase the tumor permeability or risk of metastasis. However, the increase in vasculature arising from Tinagl1 therapy reduced the expression of Hif1a significantly (p < 0.01), which may decrease the risk of drug resistance.

Recent Advances in Preclinical Research Using PAMAM Dendrimers for Cancer Gene Therapy.

Carriers of genetic material are divided into vectors of viral and non-viral origin. Viral carriers are already successfully used in experimental gene therapies, but despite advantages such as their high transfection efficiency and the wide knowledge of their practical potential, the remaining disadvantages, namely, their low capacity and complex manufacturing process, based on biological systems, are major limitations prior to their broad implementation in the clinical setting. The application of non-viral carriers in gene therapy is one of the available approaches. Poly(amidoamine) (PAMAM) dendrimers are repetitively branched, three-dimensional molecules, made of amide and amine subunits, possessing unique physiochemical properties. Surface and internal modifications improve their physicochemical properties, enabling the increase in cellular specificity and transfection efficiency and a reduction in cytotoxicity toward healthy cells. During the last 10 years of research on PAMAM dendrimers, three modification strategies have commonly been used: (1) surface modification with functional groups; (2) hybrid vector formation; (3) creation of supramolecular self-assemblies. This review describes and summarizes recent studies exploring the development of PAMAM dendrimers in anticancer gene therapies, evaluating the advantages and disadvantages of the modification approaches and the nanomedicine regulatory issues preventing their translation into the clinical setting, and highlighting important areas for further development and possible steps that seem promising in terms of development of PAMAM as a carrier of genetic material.

Ultrahigh Efficiency and Minimalist Intracellular Delivery of Macromolecules Mediated by Latent-Photothermal Surfaces.

Intracellular delivery of exogenous macromolecules by photothermal methods is still not widely employed despite its universal and clear effect on cell membrane rupture. The main causes are the unsatisfactory delivery efficiency, poor cell activity, poor cell harvest, and sophisticated operation; these challenges stem from the difficulty of simply controlling laser hotspots. Here, we constructed latent-photothermal surfaces based on multiwall carbon nanotube-doped poly(dimethyl siloxane), which can deliver cargoes with high delivery efficiency and cell viability. Also, cell release and harvest efficiencies were not affected by coordinating the hotspot content and surface structure. This system is suitable for use with a wide range of cell lines, including hard-to-transfect types. The delivery efficiency and cell viability were shown to be greater than 85 and 80%, respectively, and the cell release and harvest efficiency were greater than 95 and 80%, respectively. Moreover, this system has potential application prospects in the field of cell therapy, including stem cell neural differentiation and dendritic cell vaccines.

Intratumoral Plasmid IL12 Expands CD8+ T Cells and Induces a CXCR3 Gene Signature in Triple-negative Breast Tumors that Sensitizes Patients to Anti-PD-1 Therapy.

PURPOSE: Triple-negative breast cancer (TNBC) is an aggressive disease with limited therapeutic options. Antibodies targeting programmed cell death protein 1 (PD-1)/PD-1 ligand 1 (PD-L1) have entered the therapeutic landscape in TNBC, but only a minority of patients benefit. A way to reliably enhance immunogenicity, T-cell infiltration, and predict responsiveness is critically needed. PATIENTS AND METHODS: Using mouse models of TNBC, we evaluate immune activation and tumor targeting of intratumoral IL12 plasmid followed by electroporation (tavokinogene telseplasmid; Tavo). We further present a single-arm, prospective clinical trial of Tavo monotherapy in patients with treatment refractory, advanced TNBC (OMS-I140). Finally, we expand these findings using publicly available breast cancer and melanoma datasets. RESULTS: Single-cell RNA sequencing of murine tumors identified a CXCR3 gene signature (CXCR3-GS) following Tavo treatment associated with enhanced antigen presentation, T-cell infiltration and expansion, and PD-1/PD-L1 expression. Assessment of pretreatment and posttreatment tissue from patients confirms enrichment of this CXCR3-GS in tumors from patients that exhibited an enhancement of CD8+ T-cell infiltration following treatment. One patient, previously unresponsive to anti-PD-L1 therapy, but who exhibited an increased CXCR3-GS after Tavo treatment, went on to receive additional anti-PD-1 therapy as their immediate next treatment after OMS-I140, and demonstrated a significant clinical response. CONCLUSIONS: These data show a safe, effective intratumoral therapy that can enhance antigen presentation and recruit CD8 T cells, which are required for the antitumor efficacy. We identify a Tavo treatment-related gene signature associated with improved outcomes and conversion of nonresponsive tumors, potentially even beyond TNBC.

GALA-Modified Lipid Nanoparticles for the Targeted Delivery of Plasmid DNA to the Lungs.

This study describes the development of lipid nanoparticles (LNPs) for the efficient and selective delivery of plasmid DNA (pDNA) to the lungs. The GALA peptide was used as a ligand to target the lung endothelium and as an endosomal escape device. Transfection activity in the lungs was significantly improved when pDNA was encapsulated in double-coated LNPs. The inner coat was composed of dioleoylphsophoethanolamine and a stearylated octaarginine (STR-R8) peptide, while the outer coat was largely a cationic lipid, di-octadecenyl-trimethylammonium propane, mixed with YSK05, a pH-sensitive lipid, and cholesterol. Optimized amounts of YSK05 and GALA were used to achieve an efficient and lung-selective system. The optimized system produced a high gene expression level in the lungs (>107 RLU/mg protein) with high lung/liver and lung/spleen ratios. GALA/R8 modification and the double-coating design were indispensable for efficient gene expression in the lungs. Despite the fact that NPs prepared with 1-step or 2-step coating have the same lipid amount and composition and the same pDNA dose, the transfection activity was dramatically higher in the lungs in the case of 2-step coating. Surprisingly, 1-step or 2-step coatings had no effect on the amount of nanoparticles that were delivered to the lungs, suggesting that the double-coating strategy substantially improved the efficiency of gene expression at the intracellular level.

Protein liposomes-mediated targeted acetylcholinesterase gene delivery for effective liver cancer therapy.

BACKGROUND: Effective methods to deliver therapeutic genes to solid tumors and improve their bioavailability are the main challenges of current medical research on gene therapy. The development of efficient non-viral gene vector with tumor-targeting has very important application value in the field of cancer therapy. Proteolipid integrated with tumor-targeting potential of functional protein and excellent gene delivery performance has shown potential for targeted gene therapy. RESULTS: Herein, we prepared transferrin-modified liposomes (Tf-PL) for the targeted delivery of acetylcholinesterase (AChE) therapeutic gene to liver cancer. We found that the derived Tf-PL/AChE liposomes exhibited much higher transfection efficiency than the commercial product Lipo 2000 and shown premium targeting efficacy to liver cancer SMMC-7721 cells in vitro. In vivo, the Tf-PL/AChE could effectively target liver cancer, and significantly inhibit the growth of liver cancer xenografts grafted in nude mice by subcutaneous administration. CONCLUSIONS: This study proposed a transferrin-modified proteolipid-mediated gene delivery strategy for targeted liver cancer treatment, which has a promising potential for precise personalized cancer therapy.

Impact of a Plasmid DNA-Based Alphavirus Vaccine on Immunization Efficiency.

Alphavirus vectors have been engineered for high-level gene expression relying originally on replication-deficient recombinant particles, more recently designed for plasmid DNA-based administration. As alphavirus-based DNA vectors encode the alphavirus RNA replicon genes, enhanced transgene expression in comparison to conventional DNA plasmids is achieved. Immunization studies with alphavirus-based DNA plasmids have elicited specific antibody production, have generated tumor regression and protection against challenges with infectious agents and tumor cells in various animal models. A limited number of clinical trials have been conducted with alphavirus DNA vectors. Compared to conventional plasmid DNA-based immunization, alphavirus DNA vectors required 1000-fold less DNA to elicit similar immune responses in rodents.

Conception of Plasmid DNA and Polyethylenimine Delivery Systems with Potential Application in DNA Vaccines Field.

In DNA-based therapy research, the conception of a suitable vector to promote the target gene carriage, protection, and delivery to the cell is imperative. Exploring the interactions between polyethylenimine (PEI) and a plasmid DNA can give rise to the formation of suitable complexes for gene release and concomitant protein production. The nanosystems formulation method, based on coprecipitation, seems to be adequate for the conception of nanoparticles with suitable properties (morphology, size, surface charge, and pDNA complexation capacity) for intracellular applications. The developed systems are able of cell uptake, intracellular trafficking, and gene expression, in an extent depending on the ratio of nitrogen to phosphate groups (N/P). It comes that the transfection process can be tailored by this parameter and, therefore, also the therapeutic outcomes. This knowledge contributes for progresses in the development of suitable delivery systems with potential application in DNA vaccines field.

Inhalation delivery technology for genome-editing of respiratory diseases.

The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system has significant therapeutic potentials for lung congenital diseases such as cystic fibrosis, as well as other pulmonary disorders like lung cancer and obstructive diseases. Local administration of CRISPR/Cas9 therapeutics through inhalation can achieve high drug concentration and minimise systemic exposure. While the field is advancing with better understanding on the biological functions achieved by CRISPR/Cas9 systems, the lack of progress in inhalation formulation and delivery of the molecule may impede their clinical translation efficiently. This forward-looking review discussed the current status of formulations and delivery for inhalation of relevant biologics such as genes (plasmids and mRNAs) and proteins, emphasising on their design strategies and preparation methods. By adapting and optimising formulation strategies used for genes and proteins, we envisage that development of inhalable CRISPR/Cas9 liquid or powder formulations for inhalation administration can potentially be fast-tracked in near future.

Cellular gene delivery via poly(hexamethylene biguanide)/pDNA self-assembled nanoparticles.

Cellular gene delivery via polycations has wide implications for the potential of gene therapy, but it has remained a challenge due to the plethora of pre- and post-uptake barriers that must be overcome to reach desired efficiency. Herein we report poly(hexamethylene biguanide) (PHMB) as a nano-vector for intracellular delivery of plasmid DNA (pDNA) and oligodeoxynucleotides (ODNs). PHMB and pDNA or ODNs self-assembled into complex nanoparticles at different pH values (7.4 and 12). Their size, charge, cellular uptake, and gene-expression efficiency are assessed and compared to PEI analogues. The systematic results show that the nanoparticles are effective in delivering plasmid DNA and ODNs to model cell lines in culture (HepG2, HEK293T, HeLa), with measurable changes in gene expression levels, comparable to and, in some conditions, even higher than PEI. The well-accepted safety profile of PHMB makes it a valuable candidate for consideration as an effective intracellular DNA vector for further study and potential clinical translation.

Visualization of extracellular vesicles in the regenerating caudal fin blastema of zebrafish using in vivo electroporation.

Zebrafish have high regenerative ability in several organs including the fin. Although various mechanisms underlying fin regeneration have been revealed, some mechanisms remain to be elucidated. Recently, extracellular vesicles (EVs) have been the focus of research with regard to their role in cell-to-cell communication. It has been suggested that cells in regenerating tissues communicate using EVs. In this study, we examined the involvement of EVs in the caudal fin regeneration of zebrafish using an in vivo electroporation method. The process of regeneration appeared normal after in vivo electroporation, and the transferred plasmid showed mosaic expression in the blastema. We took advantage of this mosaic expression to observe the distribution of exosomal markers in the blastema. We transferred exosomal markers by in vivo electroporation and identified EVs in the regenerating caudal fin. The results suggest that blastemal cells communicate with other cells via EVs during caudal fin regeneration.

Nucleic Acid Delivery with α-Tocopherol-Polyethyleneimine-Polyethylene Glycol Nanocarrier System.

PURPOSE: Nucleic acid-based therapies are a promising therapeutic tool. The major obstacle in their clinical translation is their efficient delivery to the desired tissue. We developed a novel nanosized delivery system composed of conjugates of α-tocopherol, polyethyleneimine, and polyethylene glycol (TPP) to deliver nucleic acids. METHODS: We synthesized a panel of TPP molecules using different molecular weights of PEG and PEI and analyzed with various analytical approaches. The optimized version of TPP (TPP111 - the 1:1:1 molecular ratio) was self-assembled in water to produce nanostructures and then evaluated in diversified in vitro and in vivo studies. RESULTS: Through a panel of synthesized molecules, TPP111 conjugate components self-assembled in water, forming globular shaped nanostructures of ~90 nm, with high nucleic acid entrapment efficiency. The polymer had low cytotoxicity in vitro and protected nucleic acids from nucleases. Using a luciferase-expressing plasmid, TPP111-plasmid nano-complexes were rapidly up-taken by cancer cells in vitro and induced strong transfection, comparable to PEI. Colocalization of the nano-complexes and endosomes/lysosomes suggested an endosome-mediated uptake. Using a subcutaneous tumor model, intravenously injected nano-complexes preferentially accumulated to the tumor area over 24 h. CONCLUSION: These results indicate that we successfully synthesized the TPP111 nanocarrier system, which can deliver nucleic acids in vitro and in vivo and merits further evaluation.

An effective vaginal gel to deliver CRISPR/Cas9 system encapsulated in poly (ß-amino ester) nanoparticles for vaginal gene therapy.

BACKGROUND: Gene therapy has held promises for treating specific genetic diseases. However, the key to clinical application depends on effective gene delivery. METHODS: Using a large animal model, we developed two pharmaceutical formulations for gene delivery in the pigs' vagina, which were made up of poly (ß-amino ester) (PBAE)-plasmid polyplex nanoparticles (NPs) based two gel materials, modified montmorillonite (mMMT) and hectorite (HTT). FINDINGS: By conducting flow cytometry of the cervical cells, we found that PBAE-GFP-NPs-mMMT gel was more efficient than PBAE-GFP-NPs-HTT gel in delivering exogenous DNA intravaginally. Next, we designed specific CRISPR/SpCas9 sgRNAs targeting porcine endogenous retroviruses (PERVs) and evaluated the genome editing efficacy in vivo. We discovered that PERV copy number in vaginal epithelium could be significantly reduced by the local delivery of the PBAE-SpCas9/sgRNA NPs-mMMT gel. Comparable genome editing results were also obtained by high-fidelity version of SpCas9, SpCas9-HF1 and eSpCas9, in the mMMT gel. Further, we confirmed that the expression of topically delivered SpCas9 was limited to the vagina/cervix and did not diffuse to nearby organs, which was relatively safe with low toxicity. INTERPRETATION: Our data suggested that the PBAE-NPs mMMT vaginal gel is an effective preparation for local gene therapy, yielding insights into novel therapeutic approaches to sexually transmitted disease in the genital tract. FUNDING: This work was supported by the National Science and Technology Major Project of the Ministry of science and technology of China (No. 2018ZX10301402); the National Natural Science Foundation of China (81761148025, 81871473 and 81402158); Guangzhou Science and Technology Programme (No. 201704020093); National Ten Thousand Plan-Young Top Talents of China, Fundamental Research Funds for the Central Universities (17ykzd15 and 19ykyjs07); Three Big Constructions-Supercomputing Application Cultivation Projects sponsored by National Supercomputer Center In Guangzhou; the National Research FFoundation (NRF) South Africa under BRICS Multilateral Joint Call for Proposals; grant 17-54-80078 from the Russian Foundation for Basic Research.

Defining Transcription Regulatory Elements in the Human Frataxin Gene: Implications for Gene Therapy.

Friedreich's ataxia (FRDA) is the most common inherited form of ataxia in humans. It is caused by severe downregulation of frataxin (FXN) expression instigated by hyperexpansion of the GAA repeats located in intron 1 of the FXN gene. Despite numerous studies focused on identifying compounds capable of stimulating FXN expression, current knowledge regarding cis-regulatory elements involved in FXN gene expression is lacking. Using a combination of episomal and genome-integrated constructs, we defined a minimal endogenous promoter sequence required to efficiently drive FXN expression in human cells. We generated 19 constructs varying in length of the DNA sequences upstream and downstream of the ATG start codon. Using transient transfection, we evaluated the capability of these constructs to drive FXN expression. These analyses allowed us to identify a region of the gene indispensable for FXN expression. Subsequently, selected constructs containing the FXN expression control regions of varying lengths were site specifically integrated into the genome of HEK293T and human-induced pluripotent stem cells (iPSCs). FXN expression was detected in iPSCs and persisted after differentiation to neuronal and cardiac cells, indicating lineage independent function of defined regulatory DNA sequences. Finally, based on these results, we generated AAV encoding miniFXN genes and demonstrated in vivo FXN expression in mice. Results of these studies identified FXN sequences necessary to express FXN in human and mouse cells and provided rationale for potential use of endogenous FXN sequence in gene therapy strategies for FRDA.

Anti-tumor efficacy of plasmid encoding emm55 in a murine melanoma model.

Emm55 is a bacterial gene derived from Streptococcus pyogenes (S. pyogenes) that was cloned into a plasmid DNA vaccine (pAc/emm55). In this study, we investigated the anti-tumor efficacy of pAc/emm55 in a B16 murine melanoma model. Intralesional (IL) injections of pAc/emm55 significantly delayed tumor growth compared to the pAc/Empty group. There was a significant increase in the CD8+ T cells infiltrating into the tumors after pAc/emm55 treatment compared to the control group. In addition, we observed that IL injection of pAc/emm55 increased antigen-specific T cell infiltration into tumors. Depletion of CD4+ or CD8+ T cells abrogated the anti-tumor effect of pAc/emm55. Combination treatment of IL injection of pAc/emm55 with anti-PD-1 antibody significantly delayed tumor growth compared to either monotherapy. pAc/emm55 treatment combined with PD-1 blockade enhanced anti-tumor immune response and improved systemic anti-tumor immunity. Together, these strategies may lead to improvements in the treatment of patients with melanoma.

Effect of Kisspeptin-54 immunization on performance, carcass characteristics, meat quality and safety of Yiling goats.

This study aimed to evaluate the effects of kisspeptin-54 immunocastration vaccine on performance, carcass characteristics, meat quality, and safety of Yiling goats. Thirty buck goats were randomly assigned into three groups: PVAX-B2L-Kisspeptin-54-asd immunized (PBK-asd), control, and surgically castrated. PBK-asd immunization significantly stimulated serum anti-kisspeptin antibody production and reduced testosterone hormone compared with the control group (p < .05). Interestingly, PBK-asd plasmid did not integrate into the host genome and had no significant effect on growth hormone, body weight, and average daily gain (ADG). Conversely, surgical castration significantly reduced ADG and carcass weight compared to the control group. Furthermore, PBK-asd immunization did not affect carcass characteristics (dressing percentage, loin area, and fat thickness) and meat quality traits (pH, color, cooking loss, drip loss, and shearing force). These results indicate that the Kisspeptin-54 DNA vaccine is safe and has potential to be used as an alternative to surgical castration for goats without negatively affecting carcass and meat quality.