Characterization of plant produced VHH antibodies against cobra venom toxins for antivenom therapy.

Cobra (Naja kaouthia) venom contains many toxins including α-neurotoxin (αNTX) and phospholipase A2 (PLA2), which can cause neurodegeneration, respiratory failure, and even death. The traditional antivenom derived from animal serum faces many challenges and limitations. Heavy-chain-only antibodies (HCAb), fusing VHH with human IgG Fc region, offer advantages in tissue penetration, antigen binding, and extended half-life. This research involved the construction and transient expression of two types of VHH-FC which are specific to α-Neurotoxin (VHH-αNTX-FC) and Phospholipase A2 (VHH-PLA2-FC) in Nicotiana benthamiana leaves. The recombinant HCAbs were incubated for up to six days to optimize expression levels followed by purification by affinity chromatography and characterization using LC/Q-TOF mass spectrometry (MS). Purified proteins demonstrated over 92 % sequence coverage and an average mass of around 82 kDa with a high-mannose N-glycan profile. An antigen binding assay showed that the VHH-αNTX-Fc has a greater ability to bind to crude venom than VHH-PLA2-Fc.

Effect of plant produced Anti-hIL-6 receptor antibody blockade on pSTAT3 expression in human peripheral blood mononuclear cells.

As a response to invasion by pathogens, the secretion of interleukin 6 (IL-6) which is a cytokine, activates IL-6/JAKs/STAT3 intracellular signaling via., phosphorylation. Over expression of pSTAT3 induces IL-6 positive feedback loop causing cytokine release syndrome or cytokine storm. Plants have gained momentum as an alternative expression system. Hence, this study aims to produce mAb targeting human IL-6 receptor (hIL-6R) in Nicotiana benthamiana for down regulating its cellular signaling thus, decreasing the expression of pSTAT3. The variable regions of heavy and light chains of anti-hIL-6R mAb were constructed in pBYK2e geminiviral plant expression vector and transiently co-expressed in N. benthamiana. The results demonstrate the proper protein assembly of anti-hIL-6R mAb with highest expression level of 2.24 mg/g FW at 5 dpi, with a yield of 21.4 µg/g FW after purification. The purity and N-glycosylation of plant produced antibody was analyzed, including its specificity to human IL-6 receptor by ELISA. Additionally, we investigated the effect to pSTAT3 expression in human PBMC's by flow cytometry wherein, the results confirmed lower expression of pSTAT3 with increasing concentrations of plant produced anti-hIL-6R mAb. Although, further in vivo studies are key to unveil the absolute functionality of anti-hIL-6R, we hereby show the potential of the plant platform and its suitability for the production of this therapeutic antibody.

Therapeutic efficacy of plant-produced Nivolumab in transgenic C57BL/6-hPD-1 mouse implanted with MC38 colon cancer.

The therapeutic blockade of inhibitory PD-1 signaling has emerged as an effective approach for cancer immunotherapy. Nivolumab (Opdivo®), a monoclonal antibody (mAb) targeting the PD-1 immune checkpoint, is approved for treatment of several cancer indications. It functions by blocking the PD-1-mediated T-cell inhibition thus reinstating anticancer immune responses. Tremendous advances in plant biotechnology offer an alternative and economical strategy to produce therapeutic mAbs for immune-based therapies. In this study, recombinant anti-PD-1 Nivolumab was produced in Nicotiana benthamiana and the plant-produced anti-PD-1 mAb was exploited for cancer treatment in syngeneic mice model C57BL/6 mice that were used to test the antitumor efficacy of plant produced Nivolumab, along with commercial Opdivo®. C57BL/6 syngeneic mice treated with plant produced anti-PD-1 mAb exhibited reduction in the growth of established MC38 tumors. The plant produced Nivolumab treatment showed 82.9% antitumor effect in decreasing the tumor volume along with 50% tumor-free mice, whereas Opdivo® showed 90.26% reduction in volume without any tumor-free mice. Finally, plant-derived anti-PD-1 therapy was also well tolerated in tumor-bearing mice that correlated with no significant body weight changes. Overall, our plant-produced Nivolumab elicits significant inhibition of tumor growth in vivo and provides a proof-of-concept for the production of immunotherapy targeting PD-1.

Harnessing the advances of genetic engineering in microalgae for the production of cannabinoids.

Cannabis is widely recognized as a medicinal plant owing to bioactive cannabinoids. However, it is still considered a narcotic plant, making it hard to be accessed. Since the biosynthetic pathway of cannabinoids is disclosed, biotechnological methods can be employed to produce cannabinoids in heterologous systems. This would pave the way toward biosynthesizing any cannabinoid compound of interest, especially minor substances that are less produced by a plant but have a high medicinal value. In this context, microalgae have attracted increasing scientific interest given their unique potential for biopharmaceutical production. In the present review, the current knowledge on cannabinoid production in different hosts is summarized and the biotechnological potential of microalgae as an emerging platform for synthetic production is put in perspective. A critical survey of genetic requirements and various transformation approaches are also discussed.

Harnessing the Potential of Plant Expression System towards the Production of Vaccines for the Prevention of Human Papillomavirus and Cervical Cancer.

Cervical cancer is the most common gynecological malignant tumor worldwide, and it remains a major health problem among women, especially in developing countries. Despite the significant research efforts employed for tumor prevention, cervical cancer ranks as the leading cause of cancer death. Human papillomavirus (HPV) is the most important risk factor for cervical cancer. Cervical cancer is a preventable disease, for which early detection could increase survival rates. Immunotherapies represent a promising approach in the treatment of cancer, and several potential candidates are in clinical trials, while some are available in the market. However, equal access to available HPV vaccines is limited due to their high cost, which remains a global challenge for cervical cancer prevention. The implementation of screening programs, disease control systems, and medical advancement in developed countries reduce the serious complications associated with the disease somewhat; however, the incidence and prevalence of cervical cancer in low-income and middle-income countries continues to gradually increase, making it the leading cause of mortality, largely due to the unaffordable and inaccessible anti-cancer therapeutic options. In recent years, plants have been considered as a cost-effective production system for the development of vaccines, therapeutics, and other biopharmaceuticals. Several proof-of-concept studies showed the possibility of producing recombinant biopharmaceuticals for cancer immunotherapy in a plant platform. This review summarizes the current knowledge and therapeutic options for the prevention of cervical cancer and discusses the potential of the plant expression platform to produce affordable HPV vaccines.

Feasibility of plant-expression system for production of recombinant anti-human IgE: An alternative production platform for therapeutic monoclonal antibodies.

Omalizumab, the anti-immunoglobulin IgE antibody is the only approved and available monoclonal antibody as an auxiliary medicament for the severe respiratory allergic reactions. It forms small size immune complexes by binding to free IgE, thereby inhibiting the interaction of IgE with its receptors. Additionally, the anti-IgE can also differently shape the airflow by impeding the stimulation of IgE receptors present on structural cells in the respiratory tract. The present study aimed to use plants as an expression system for anti-human IgE antibody production, using Nicotiana benthamiana as hosts. Recombinant Agrobacterium tumefaciens containing heavy chain (HC) and light chain (LC) domains of anti-human IgE were co-transformed in N. benthamiana. The assembling of the antibody and its expression was detected by SDS-PAGE and Western blot analysis. The functional ability of the anti-IgE antibody was determined via its binding capacity with target IgE by ELISA and the inhibition of basophil activation. The anti-human IgE mAb generated in plants was shown to be effective in binding to its target IgE and inhibit the IgE-crosslink in RS-ATL8 reporter cells. Although, antibody yield and purification process have to be further optimized, this study demonstrates the use of plant expression system as a promising platform for the production of Omalizumab which showed a comparable in vitro function to that of commercial Omalizumab (Xolair) in the inhibition of basophil activation.

Expression of plant-produced anti-PD-L1 antibody with anoikis sensitizing activity in human lung cancer cells via., suppression on epithelial-mesenchymal transition.

Immune checkpoint antibodies in cancer treatment are receptor-ligand pairs that modulate cancer immunity. PD-1/PD-L1 pathway has emerged as one of the major targets in cancer immunotherapy. Atezolizumab, the first anti-PD-L1 antibody approved for the treatment of metastatic urothelial, non-small cell lung, small cell lung and triple-negative breast cancers, is produced in Chinese Hamster Ovary (CHO) cells with several limitations i.e., high-production costs, low-capacity yields, and contamination risks. Due to the rapid scalability and low production costs, the transient expression in Nicotiana benthamiana leaves was investigated by co-infiltration of Agrobacterium tumefaciens GV3101 cultures harboring the nucleic acid sequences encoding for Atezolizumab heavy chain and light chain in this study. The transient expression of Atezolizumab in transformed N. benthamiana accumulated up to 86.76 µg/g fresh leaf weight after 6 days of agroinfiltration (OD 600 nm: 0.4) with 1:1 ratio of heavy chain to light chain. The structural and functional characteristics of plant-produced Atezolizumab was compared with commercially available Tecentriq® from CHO cells with similar binding efficacies to PD-L1 receptor. The direct anti-cancer effect of plant-produced anti-PD-L1 was further performed in human lung metastatic cancer cells H460 cultured under detachment condition, demonstrating the activity of anti-PD-L1-antibody on sensitizing anoikis as well as the suppression on anti-apoptosis proteins (Bcl-2 and Mcl-1) and modulation of epithelial to mesenchymal regulating proteins (E-cadherin, N-cadherin, Snail and Slug). In conclusion, this study manifests plants as an alternative cost-effective platform for the production of functional monoclonal antibodies for use in cancer therapy.

An Algae-Made RBD from SARS-CoV-2 Is Immunogenic in Mice.

Despite the current advances in global vaccination against SARS-CoV-2, boosting is still required to sustain immunity in the population, and the induction of sterilizing immunity remains as a pending goal. Low-cost oral immunogens could be used as the basis for the design of affordable and easy-to-administer booster vaccines. Algae stand as promising platforms to produce immunogens at low cost, and it is possible to use them as oral delivery carriers since they are edible (not requiring complex purification and formulation processes). Herein, a Chlamydomonas-made SARS-CoV-2 RBD was evaluated as an oral immunogen in mice to explore the feasibility of developing an oral algae-based vaccine. The test immunogen was stable in freeze-dried algae biomass and able to induce, by the oral route, systemic and mucosal humoral responses against the spike protein at a similar magnitude to those induced by injected antigen plus alum adjuvant. IgG subclass analysis revealed a Th2-bias response which lasted over 4 months after the last immunization. The induced antibodies showed a similar reactivity against either Delta or Omicron variants. This study represents a step forward in the development of oral vaccines that could accelerate massive immunization.

Preclinical evaluation of a plant-derived SARS-CoV-2 subunit vaccine: Protective efficacy, immunogenicity, safety, and toxicity.

Coronavirus disease 2019 (COVID-19) is an acute respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The prevention of SARS-CoV-2 transmission has become a global priority. Previously, we showed that a protein subunit vaccine that was developed based on the fusion of the SARS-CoV-2 receptor-binding domain (RBD) to the Fc portion of human IgG1 (RBD-Fc), produced in Nicotiana benthamiana, and adjuvanted with alum, namely, Baiya SARS-CoV-2 Vax 1, induced potent immunological responses in both mice and cynomolgus monkeys. Hence, this study evaluated the protective efficacy, safety, and toxicity of Baiya SARS-CoV-2 Vax 1 in K18-hACE2 mice, monkeys and Wistar rats. Two doses of vaccine were administered three weeks apart on Days 0 and 21. The administration of the vaccine to K18-hACE2 mice reduced viral loads in the lungs and brains of the vaccinated animals and protected the mice against challenge with SARS-CoV-2. In monkeys, the results of safety pharmacology tests, general clinical observations, and a core battery of studies of three vital systems, namely, the central nervous, cardiovascular, and respiratory systems, did not reveal any safety concerns. The toxicology study of the vaccine in rats showed no vaccine-related pathological changes, and all the animals remained healthy under the conditions of this study. Furthermore, the vaccine did not cause any abnormal toxicity in rats and was clinically tolerated even at the highest tested concentration. In addition, general health status, body temperature, local toxicity at the administration site, hematology, and blood chemistry parameters were also monitored. Overall, this work presents the results of the first systematic study of the safety profile of a plant-derived vaccine, Baiya SARS-CoV-2 Vax 1; this approach can be considered a viable strategy for the development of vaccines against COVID-19.

Production of Genistein in Amaranthus tricolor var. tristis and Spinacia oleracea by Expression of Glycine max Isoflavone Synthase.

Isoflavonoids, the diverse group of secondary metabolites derived from the phenylpropanoid pathway, are distributed predominantly in leguminous plants. It has received considerable attention in recent days due to its health promoting benefits and is known to prevent certain diseases in humans. These isoflavonoids are synthesized from flavonoid intermediates of phenylpropanoid pathway by the enzyme isoflavone synthase. Metabolic engineering of isoflavonoid biosynthesis in non-legume crop plants could offer the health benefits of these compounds in diverse plant species further contributing for crop improvement. The transient expression of heterologous genes in the host is considered as an alternative to stable expression, that can provide a rapid way of studying the pathway engineering for metabolite production and could also act as a production platform for nutraceuticals and biopharmaceuticals. In this study, isoflavone genistein was produced in Amaranthus tricolor var. tristis and Spinacia oleracea by transiently expressing Glycine max isoflavone synthase (GmIFS). The GmIFS gene was cloned in plant expression vector pEarleyGate 102 HA and pEAQ-HT-DEST 3 and transformed into plants by agroinfiltration. The presence of transgene in the agroinfiltrated leaves was confirmed by semiquantitative reverse-transcription polymerase chain reaction. The flavonoid substrate naringenin and isoflavonoid genistein were quantified using high performance liquid chromatography in both wild-type and infiltrated leaf samples of both the plants. The naringenin content varied in the range of 65.5-338.5 nM/g fresh weight, while the accumulation of genistein was observed with varying concentrations from 113 to 182.6 nM/g fresh weight in the agroinfiltrated leaf samples of both A. tricolor var. tristis and S. oleracea. These results indicate that the transient expression of GmIFS gene has led to the synthesis of isoflavonoid genistein in A. tricolor var. tristis and S. oleracea providing an insight that stable expression of this gene could enrich the nutraceutical content in the crop plants. To the best of our knowledge, this is the first report on transient expression of GmIFS gene for the production of genistein in A. tricolor var. tristis and S. oleracea.

Potential for Developing Plant-Derived Candidate Vaccines and Biologics against Emerging Coronavirus Infections.

The emerging human coronavirus infections in the 21st century remain a major public health crisis causing worldwide impact and challenging the global health care system. The virus is circulating in several zoonotic hosts and continuously evolving, causing occasional outbreaks due to spill-over events occurring between animals and humans. Hence, the development of effective vaccines or therapeutic interventions is the current global priority in order to reduce disease severity, frequent outbreaks, and to prevent future infections. Vaccine development for newly emerging pathogens takes a long time, which hinders rapid immunization programs. The concept of plant-based pharmaceuticals can be readily applied to meet the recombinant protein demand by means of transient expression. Plants are evolved as an expression platform, and they bring a combination of unique interests in terms of rapid scalability, flexibility, and economy for industrial-scale production of effective vaccines, diagnostic reagents, and other biopharmaceuticals. Plants offer safe biologics to fulfill emergency demands, especially during pandemic situations or outbreaks caused by emerging strains. This review highlights the features of a plant expression platform for producing recombinant biopharmaceuticals to combat coronavirus infections with emphasis on COVID-19 vaccine and biologics development.

Biotechnological Insights on the Expression and Production of Antimicrobial Peptides in Plants.

The emergence of drug-resistant pathogens poses a serious critical threat to global public health and requires immediate action. Antimicrobial peptides (AMPs) are a class of short peptides ubiquitously found in all living forms, including plants, insects, mammals, microorganisms and play a significant role in host innate immune system. These peptides are considered as promising candidates to treat microbial infections due to its distinct advantages over conventional antibiotics. Given their potent broad spectrum of antimicrobial action, several AMPs are currently being evaluated in preclinical/clinical trials. However, large quantities of highly purified AMPs are vital for basic research and clinical settings which is still a major bottleneck hindering its application. This can be overcome by genetic engineering approaches to produce sufficient amount of diverse peptides in heterologous host systems. Recently plants are considered as potential alternatives to conventional protein production systems such as microbial and mammalian platforms due to their unique advantages such as rapidity, scalability and safety. In addition, AMPs can also be utilized for development of novel approaches for plant protection thereby increasing the crop yield. Hence, in order to provide a spotlight for the expression of AMP in plants for both clinical or agricultural use, the present review presents the importance of AMPs and efforts aimed at producing recombinant AMPs in plants for molecular farming and plant protection so far.

Insights into antioxidant activities and anti-skin-aging potential of callus extract from Centella asiatica (L.).

Formation of oxidative stress in dermal fibroblasts plays crucial roles in aging processes of skin. The use of phytochemicals that can promote capacity of fibroblasts to combat oxidative stress is an attractive strategy to prevent skin aging and promote skin beauty. Centella asiatica has been used to treat multitude of diseases for centuries. Previous investigations demonstrated that extracts from C. asiatica have a broad range of beneficial activities through their antioxidant activity. Hence, the extract from this medicinal plant could be a great candidate for anti-skin-aging agent. Callus culture offers a powerful platform for sustainable, rapid and large-scale production of phytochemicals to serve extensive demands of pharmaceutical and cosmeceutical industries. Here, we demonstrated the application of callus culture of Centella asiatica to produce bioactive metabolites. The 50% ethanolic extract of callus culture has distinctive features of chemical compositions and biological profiles. Information from HPTLC-DPPH and HPLC analysis suggested that the callus extract comprises distinctive antioxidant compounds, compared with those isolated from authentic plant. Moreover, results from cell culture experiment demonstrated that callus extract possesses promising antioxidant and anti-skin-aging activities. Pre-treatment with callus extract attenuated H2O2-induced-cytotoxicity on human dermal fibroblasts. The results from RT-qPCR clearly suggested that the upregulation of cellular antioxidant enzymes appeared to be major contributor for the protective effects of callus extract against oxidative stress. Moreover, supplementation with callus extract inhibited induction of matrix metalloprotease-9 following H2O2 exposure, suggesting its potential anti-skin-aging activity. Our results demonstrate the potential utility of C. asiatica callus extract as anti-skin-aging agent in cosmeceutical preparations.

Efficient Transient Expression of Recombinant Proteins Using DNA Viral Vectors in Freshwater Microalgal Species.

The increase in the world population, the advent of new infections and health issues, and the scarcity of natural biological products have spotlighted the importance of recombinant protein technology and its large-scale production in a cost-effective manner. Microalgae have become a significant promising platform with the potential to meet the increasing demand for recombinant proteins and other biologicals. Microalgae are safe organisms that can grow rapidly and are easily cultivated with basic nutrient requirements. Although continuous efforts have led to considerable progress in the algae genetic engineering field, there are still many hurdles to overcome before these microorganisms emerge as a mature expression system. Hence, there is a need to develop efficient expression approaches to exploit microalgae for the production of recombinant proteins at convenient yields. This study aimed to test the ability of the DNA geminiviral vector with Rep-mediated replication to transiently express recombinant proteins in the freshwater microalgal species Chlamydomonas reinhardtii and Chlorella vulgaris using Agrobacterium-mediated transformation. The SARS-CoV-2 receptor binding domain (RBD) and basic fibroblast growth factor (bFGF) are representative antigen proteins and growth factor proteins, respectively, that were subcloned in a geminiviral vector and were used for nuclear transformation to transiently express these proteins in C. reinhardtii and C. vulgaris. The results showed that the geminiviral vector allowed the expression of both recombinant proteins in both algal species, with yields at 48 h posttransformation of up to 1.14 µg/g RBD and 1.61 ng/g FGF in C. vulgaris and 1.61 µg/g RBD and 1.025 ng/g FGF in C. reinhardtii. Thus, this study provides a proof of concept for the use of DNA viral vectors for the simple, rapid, and efficient production of recombinant proteins that repress the difficulties faced in the genetic transformation of these unicellular green microalgae. This concept opens an avenue to explore and optimize green microalgae as an ideal economically valuable platform for the production of therapeutic and industrially relevant recombinant proteins in shorter time periods with significant yields.

The Potential of Algal Biotechnology to Produce Antiviral Compounds and Biopharmaceuticals.

The emergence of the Coronavirus Disease 2019 (COVID-19) caused by the SARS-CoV-2 virus has led to an unprecedented pandemic, which demands urgent development of antiviral drugs and antibodies; as well as prophylactic approaches, namely vaccines. Algae biotechnology has much to offer in this scenario given the diversity of such organisms, which are a valuable source of antiviral and anti-inflammatory compounds that can also be used to produce vaccines and antibodies. Antivirals with possible activity against SARS-CoV-2 are summarized, based on previously reported activity against Coronaviruses or other enveloped or respiratory viruses. Moreover, the potential of algae-derived anti-inflammatory compounds to treat severe cases of COVID-19 is contemplated. The scenario of producing biopharmaceuticals in recombinant algae is presented and the cases of algae-made vaccines targeting viral diseases is highlighted as valuable references for the development of anti-SARS-CoV-2 vaccines. Successful cases in the production of functional antibodies are described. Perspectives on how specific algae species and genetic engineering techniques can be applied for the production of anti-viral compounds antibodies and vaccines against SARS-CoV-2 are provided.

Emergence of Novel Coronavirus 2019-nCoV: Need for Rapid Vaccine and Biologics Development.

Novel Coronavirus (2019-nCoV) is an emerging pathogen that was first identified in Wuhan, China in late December 2019. This virus is responsible for the ongoing outbreak that causes severe respiratory illness and pneumonia-like infection in humans. Due to the increasing number of cases in China and outside China, the WHO declared coronavirus as a global health emergency. Nearly 35,000 cases were reported and at least 24 other countries or territories have reported coronavirus cases as early on as February. Inter-human transmission was reported in a few countries, including the United States. Neither an effective anti-viral nor a vaccine is currently available to treat this infection. As the virus is a newly emerging pathogen, many questions remain unanswered regarding the virus's reservoirs, pathogenesis, transmissibility, and much more is unknown. The collaborative efforts of researchers are needed to fill the knowledge gaps about this new virus, to develop the proper diagnostic tools, and effective treatment to combat this infection. Recent advancements in plant biotechnology proved that plants have the ability to produce vaccines or biopharmaceuticals rapidly in a short time. In this review, the outbreak of 2019-nCoV in China, the need for rapid vaccine development, and the potential of a plant system for biopharmaceutical development are discussed.

Metabolic Engineering of Isoflavonoid Biosynthesis by Expressing Glycine max Isoflavone Synthase in Allium cepa L. for Genistein Production.

Isoflavonoids, the diverse group of secondary metabolites derived from the phenylpropanoid pathway, are distributed predominantly in leguminous plants and play a vital role in promoting human health. Genetic engineering of the metabolite synthesis pathway has turned out to be an attractive approach for the production of various secondary metabolites. In our study, we attempted to produce the isoflavone genistein, a well-known health-promoting metabolite, in Allium cepa L. (onion) by introducing Glycine max Isoflavone synthase (GmIFS). The GmIFS gene was cloned into the pEarleyGate 102 HA vector and transformed into onion by Agrobacterium-mediated and biolistic methods. The presence of GmIFS in transgenic onion was confirmed by PCR, dot blot, and Southern hybridization. Analysis of the transgenic onion calli lines demonstrated that the expression of the GmIFS gene led to the production of isoflavone genistein in in vitro tissues. The biolistic stable transformed calli with transformation efficiency of 73% (62.65 nM/g FW) accumulated more genistein than the Agrobacterium stable transformed calli with transformation efficiency of 56% (42.5 nM/g FW). Overall, heterologous gene expression of GmIFS was demonstrated by modifying the secondary metabolite pathway in onion tissues for the production of isoflavone genistein that can boost up human health with its health-promoting properties.

Regeneration of Leydig cells in ectopically autografted adult mouse testes.

Ectopic autografting of testis tissue is a promising approach for studying testicular development, male germline preservation and restoration of male fertility. In this study, we examined the fate of various testicular cells in adult mouse testes following ectopic autografting at 1, 2, 4 and 8 weeks post grafting. Histological examination showed no evidence of re-establishment of spermatogenesis in autografts, and progressive degeneration of seminiferous tubules was detected. Expression of germ cell-specific proteins such as POU5F1, DAZL, TNP1, TNP2, PRM1 and PRM2 revealed that, although proliferating and differentiating spermatogenic germ cells such as spermatogonia, spermatocytes and spermatids could survive in autografts until 4 weeks, only terminally differentiated germ cells such as sperm persisted in autografts until 8 weeks. The presence of Sertoli and peritubular myoid cells, as indicated by expression of WT1 and ACTA2 proteins, respectively, was evident in the autografts until 8 weeks. Interestingly, seminal vesicle weight and serum testosterone level were restored in autografted mice by 8 weeks post grafting. The expression of Leydig cell-specific proteins such as CYP11A1, HSD3B2 and LHCGR showed revival of Leydig cell (LC) populations in autografts over time since grafting. Elevated expression of PDGFRA, LIF, DHH and NEFH in autografts indicated de novo regeneration of LC populations. Autografted adult testis can be used as a model for investigating Leydig cell regeneration, steroidogenesis and regulation of the intrinsic factors involved in Leydig cell development. The success of this rodent model can have therapeutic applications for adult human males undergoing sterilizing cancer therapy.