Identification of hyperosmotic stress-responsive genes in Chinese hamster ovary cells via genome-wide virus-free CRISPR/Cas9 screening.

Chinese hamster ovary (CHO) cells are the preferred mammalian host cells for therapeutic protein production that have been extensively engineered to possess the desired attributes for high-yield protein production. However, empirical approaches for identifying novel engineering targets are laborious and time-consuming. Here, we established a genome-wide CRISPR/Cas9 screening platform for CHO-K1 cells with 111,651 guide RNAs (gRNAs) targeting 21,585 genes using a virus-free recombinase-mediated cassette exchange-based gRNA integration method. Using this platform, we performed a positive selection screening under hyperosmotic stress conditions and identified 180 genes whose perturbations conferred resistance to hyperosmotic stress in CHO cells. Functional enrichment analysis identified hyperosmotic stress responsive gene clusters, such as tRNA wobble uridine modification and signaling pathways associated with cell cycle arrest. Furthermore, we validated 32 top-scoring candidates and observed a high rate of hit confirmation, demonstrating the potential of the screening platform. Knockout of the novel target genes, Zfr and Pnp, in monoclonal antibody (mAb)-producing recombinant CHO (rCHO) cells and bispecific antibody (bsAb)-producing rCHO cells enhanced their resistance to hyperosmotic stress, thereby improving mAb and bsAb production. Overall, the collective findings demonstrate the value of the screening platform as a powerful tool to investigate the functions of genes associated with hyperosmotic stress and to discover novel targets for rational cell engineering on a genome-wide scale in CHO cells.

Enhancing CHO cell productivity through a dual selection system using Aspg and Gs in glutamine free medium.

The dominant method for generating Chinese hamster ovary (CHO) cell lines that produce high titers of biotherapeutic proteins utilizes selectable markers such as dihydrofolate reductase (Dhfr) or glutamine synthetase (Gs), alongside inhibitory compounds like methotrexate or methionine sulfoximine, respectively. Recent work has shown the importance of asparaginase (Aspg) for growth in media lacking glutamine-the selection medium for Gs-based selection systems. We generated a Gs/Aspg double knockout CHO cell line and evaluated its utility as a novel dual selectable system via co-transfection of Gs-Enbrel and Aspg-Enbrel plasmids. Using the same selection conditions as the standard Gs system, the resulting cells from the Gs/Aspg dual selection showed substantially improved specific productivity and titer compared to the standard Gs selection method, however, with reduced growth rate and viability. Following adaptation in the selection medium, the cells improved viability and growth while still achieving ~5-fold higher specific productivity and ~3-fold higher titer than Gs selection alone. We anticipate that with further optimization of culture medium and selection conditions, this approach would serve as an effective addition to workflows for the industrial production of recombinant biotherapeutics.

A metabolic CRISPR-Cas9 screen in Chinese hamster ovary cells identifies glutamine-sensitive genes.

Media and feed optimization have fueled many-fold improvements in mammalian biopharmaceutical production, but genome editing offers an emerging avenue for further enhancing cell metabolism and bioproduction. However, the complexity of metabolism, involving thousands of genes, makes it unclear which engineering strategies will result in desired traits. Here we present a comprehensive pooled CRISPR screen for CHO cell metabolism, including ~16,000 gRNAs against ~2500 metabolic enzymes and regulators. Using this screen, we identified a glutamine response network in CHO cells. Glutamine is particularly important since it is often over-fed to drive increased TCA cycle flux, but toxic ammonia may accumulate. With the screen we found one orphan glutamine-responsive gene with no clear connection to our network. Knockout of this novel and poorly characterized lipase, Abhd11, substantially increased growth in glutamine-free media by altering the regulation of the TCA cycle. Thus, the screen provides an invaluable targeted platform to comprehensively study genes involved in any metabolic trait, and elucidate novel regulators of metabolism.

Awakening dormant glycosyltransferases in CHO cells with CRISPRa.

Chinese hamster ovary (CHO) cells are the preferred workhorse for the biopharmaceutical industry, and CRISPR/Cas9 has proven powerful for generating targeted gene perturbations in CHO cells. Here, we expand the CRISPR engineering toolbox with CRISPR activation (CRISPRa) to increase transcription of endogenous genes. We successfully increased transcription of Mgat3 and St6gal1, and verified their activity on a functional level by subsequently detecting that the appropriate glycan structures were produced. This study demonstrates that CRISPRa can make targeted alterations of CHO cells for desired phenotypes.

Reduced apoptosis in Chinese hamster ovary cells via optimized CRISPR interference.

Chinese hamster ovary (CHO) cells are widely used for biopharmaceutical protein production. One challenge limiting CHO cell productivity is apoptosis stemming from cellular stress during protein production. Here we applied CRISPR interference (CRISPRi) to downregulate the endogenous expression of apoptotic genes Bak, Bax, and Casp3 in CHO cells. In addition to reduced apoptosis, mitochondrial membrane integrity was improved and the caspase activity was reduced. Moreover, we optimized the CRISPRi system to enhance the gene repression efficiency in CHO cells by testing different repressor fusion types. An improved Cas9 repressor has been identified by applying C-terminal fusion of a bipartite repressor domain, KRAB-MeCP2, to nuclease-deficient Cas9. These results collectively demonstrate that CHO cells can be rescued from cell apoptosis by targeted gene repression using the CRISPRi system.

Laser-Assisted Photodynamic Therapy: Two Novel Protocols for Enhanced Treatment Results.

Photodynamic therapy (PDT) uses a topical photosensitizing agent which is activated by a light source to cause destruction of specific cells. Commonly used for the treatment of actinic keratoses and photodamage, PDT can also be used for other conditions including acne and sebaceous hyperplasia. Here we report our experience with two treatment protocols. The first protocol utilizes laser assisted delivery of topical 5-aminolevulinic acid for enhanced efficacy of blue light photodynamic therapy in the treatment of actinic keratoses and photodamage. The second protocol utilizes red light photodynamic therapy followed by pulsed dye laser to effectively target sebaceous glands in patients with extensive sebaceous hyperplasia.

J Drugs Dermatol. 2017;16(4):329-331.

Development and characterization of ceramic-polymeric hybrid scaffolds for bone regeneration: incorporating of bioactive glass BG-58S into PDLLA matrix.

In recent years, there has been a notable surge of interest in hybrid materials within the biomedical field, particularly for applications in bone repair and regeneration. Ceramic-polymeric hybrid scaffolds have shown promising outcomes. This study aimed to synthesize bioactive glass (BG-58S) for integration into a bioresorbable polymeric matrix based on PDLLA, aiming to create a bioactive scaffold featuring stable pH levels. The synthesis involved a thermally induced phase separation process followed by lyophilization to ensure an appropriate porous structure. BG-58S characterization revealed vitreous, bioactive, and mesoporous structural properties. The scaffolds were analyzed for morphology, interconnectivity, chemical groups, porosity and pore size distribution, zeta potential, pH, in vitro degradation, as well as cell viability tests, total protein content and mineralization nodule production. The PDLLA scaffold displayed a homogeneous morphology with interconnected macropores, while the hybrid scaffold exhibited a heterogeneous morphology with smaller diameter pores due to BG-58S filling. The hybrid scaffold also demonstrated a pH buffering effect on the polymer surface. In addition to structural characteristics, degradation tests indicated that by incorporating BG-58S modified the acidic degradation of the polymer, allowing for increased total protein production and the formation of mineralization nodules, indicating a positive influence on cell culture.

Multiplex genome editing eliminates the Warburg Effect without impacting growth rate in mammalian cells.

The Warburg effect is ubiquitous in proliferative mammalian cells, including cancer cells, but poses challenges for biopharmaceutical production, as lactate accumulation inhibits cell growth and protein production. Previous efforts to eliminate lactate production via knockout have failed in mammalian bioprocessing since lactate dehydrogenase has proven essential. However, here we eliminated the Warburg effect in Chinese hamster ovary (CHO) and HEK293 cells by simultaneously knocking out lactate dehydrogenase and regulators involved in a negative feedback loop that typically inhibits pyruvate conversion to acetyl-CoA. In contrast to long-standing assumptions about the role of aerobic glycolysis, Warburg-null cells maintain wildtype growth rate while producing negligible lactate. Further characterization of Warburg-null CHO cells showed a compensatory increase in oxygen consumption, a near total reliance on oxidative metabolism, and higher cell densities in fed-batch cell culture. These cells remained amenable for production of diverse biotherapeutic proteins, reaching industrially relevant titers and maintaining product glycosylation. Thus, the ability to eliminate the Warburg effect is an important development for biotherapeutic production and provides a tool for investigating a near-universal metabolic phenomenon.

1927-nm fractional resurfacing of facial actinic keratoses: a promising new therapeutic option.

BACKGROUND: Actinic keratoses (AK) are precancerous epidermal proliferations commonly present on chronically sun-damaged skin. These lesions are among the most often treated dermatologic conditions. OBJECTIVE: We sought to investigate the 6-month safety, tolerance, and efficacy of nonablative 1927-nm fractional resurfacing of facial AK. METHODS: This was a prospective clinical trial of 24 individuals with facial photodamage and AK receiving up to 4 treatments with the fractionated 1927-nm nonablative thulium laser. RESULTS: At 6 months, an 86.6% reduction in absolute number of lesions was noted by independent physician assessment. In addition, at this same time point, patients reported marked or noticeable improvement in overall photodamage. LIMITATIONS: This prospective study does not provide safety, tolerance, and efficacy data beyond 6 months of follow-up, nor does it identify the precise mechanism of action involved in AK clearance after 1927-nm resurfacing. CONCLUSION: The clinical and histologic findings, as well as the reported patient satisfaction and safety, suggest that the treatment of AK and photodamage with a fractionated 1927-nm nonablative thulium laser is a promising new therapeutic option.

Topical perfluorodecalin resolves immediate whitening reactions and allows rapid effective multiple pass treatment of tattoos.

BACKGROUND AND OBJECTIVE: Laser tattoo removal using multiple passes per session, with each pass delivered after spontaneous resolution of whitening, improves tattoo fading in a 60-minute treatment time. Our objective was to evaluate the safety and efficacy of topical perfluorodecalin (PFD) in facilitating rapid effective multiple-pass tattoo removal. STUDY DESIGN: In a randomized, controlled study using Q-switched ruby or Nd:YAG laser, 22 previously treated tattoos were treated with 3 passes using PFD to resolve whitening after each pass ("R0 method"). In previously untreated symmetric tattoos, seven were treated over half of the tattoo with the R20 method, and the opposite half with 4 passes using PFD (R0 method); two were treated over half with a single pass and the opposite half with 4 passes using PFD (R0 method); and six treated over half with a single pass followed by PFD and the opposite half with a single pass alone. Blinded dermatologists rated tattoo fading at 1-3 months. Optical coherence tomography (OCT) imaging of whitening was performed in two tattoos. RESULTS: Topical PFD clinically resolved immediate whitening reactions within a mean 5 seconds (range 3-10 seconds). Tattoos treated with the R0 method demonstrated excellent fading in an average total treatment time of 5 minutes. Tattoo areas treated with the R0 method demonstrated equal fading compared to the R20 method, and improved fading compared to a single pass method. OCT imaging of whitening demonstrated epidermal and dermal hyper-reflective "bubbles" that dissipated until absent at 9-10 minutes after PFD application, and at 20 minutes without intervention. CONCLUSIONS: Multiple-pass tattoo removal using PFD to deliver rapid sequential passes (R0 method) appears equally effective as the R20 method, in a total treatment time averaging 5 minutes, and more effective than single pass treatment. OCT-visualized whitening-associated "bubbles," upon treatment with PFD, resolve twice as rapidly as spontaneous resolution.

Retrospective study of the treatment of infantile hemangiomas using a combination of propranolol and pulsed dye laser.

BACKGROUND: Infantile hemangioma (IH) clearance may be slow or incomplete in response to pulsed dye laser (PDL) or propranolol alone. OBJECTIVES: To evaluate whether IH treated with PDL and propranolol displayed more rapid and complete clearance than IH treated with propranolol alone. MATERIALS AND METHODS: Retrospective review of facial-segmental IH treated with propranolol and PDL and controls treated with propranolol was conducted. Blinded physicians used patient photographs to select clearance level and the earliest date of near-complete clearance. Days of propranolol, PDL sessions, and propranolol dose, each until date of near-complete clearance; total days of propranolol; and total propranolol dose were recorded. RESULTS: Infantile hemangiomas treated concurrently with propranolol and PDL achieved complete clearance (6/12) more often than IH treated with propranolol followed by PDL (2/5) or IH treated with propranolol alone (1/8; difference in clearance scores p = .01) and achieved near-complete clearance after fewer days of propranolol (mean 92 days for concurrent propranolol and PDL vs 288 days for propranolol; p < .001). Cumulative propranolol dose until near-complete clearance was lowest in the concurrent propranolol and PDL group (149.16 vs. 401.25 mg/kg for propranolol; p < .001). CONCLUSION: Facial-segmental IH treated with propranolol and PDL displayed morerapid and complete clearance and required a lower cumulative propranolol dose to achieve near-complete clearance.

Treatment of port-wine stains with a short pulse width 532-nm Nd:YAG laser.

BACKGROUND AND OBJECTIVE: Pulsed dye laser treatment often results in port-wine stain (PWS) improvement; however, results vary. A frequency-doubled neodymium-doped yttrium aluminum garnet (Nd:YAG) laser that allows for shorter pulse widths along with large spot sizes and high fluences has been developed for the treatment of cutaneous vascular lesions. STUDY DESIGN: A prospective, controlled study was performed in 5 adults with PWS using a frequency-doubled Nd:YAG laser (Excel V; Cutera Inc, Brisbane, CA) in 4 quadrants, using spot sizes of 6 to 10 mm, fluences of 4.8 to 9 J/cm2, and pulse durations of 3 to 6 ms. An adjacent control area was not treated. Each was assessed immediately posttreatment for purpura and edema and at 1 month for PWS color, size, texture, and thickness. Skin biopsies obtained immediately after and at 1 month posttreatment were evaluated. RESULTS: All treatment quadrants displayed purpura. At 1-month follow-up, all treatment quadrants showed at least 1 grade of color improvement, from a minimum of 1% to 25% to a maximum of 51% to 75% improvement (12/20 quadrants with 1%-25% improvement, 3/20 with 26%-50%, 5/20 with 51%-75%, and 0/20 with 76%-100%). Histologic evaluation of treatment quadrants revealed vascular changes ranging 0.35 to 4 mm in depth. Immediately posttreatment, thrombi and extravasated red blood cells were observed in treatment quadrants. Histology at 1 month revealed decreased number and diameter of vessels in treatment quadrants (superficial vessels decreased by mean 1.1 vessels per section [13%], and diameter by 3.0 µm [47%], midlevel vessels decreased in number by 2.3 [20%], diameter by 2.42 µm [25%], and deep vessels decreased in number by 1.5 [83%], and diameter by 7.44 µm [88%]). CONCLUSIONS: A single treatment with a short pulse width, frequency-doubled Nd:YAG laser resulted in safe and effective improvement of PWS, with up to 75% improvement in color observed at 1 month. Histologic evaluation demonstrated vascular injury at depths of 0.35 to 4 mm with a reduction in vessel number and size at multiple dermal levels.

From observational to actionable: rethinking omics in biologics production.

As the era of omics continues to expand with increasing ubiquity and success in both academia and industry, omics-based experiments are becoming commonplace in industrial biotechnology, including efforts to develop novel solutions in bioprocess optimization and cell line development. Omic technologies provide particularly valuable 'observational' insights for discovery science, especially in academic research and industrial R&D; however, biomanufacturing requires a different paradigm to unlock 'actionable' insights from omics. Here, we argue the value of omic experiments in biotechnology can be maximized with deliberate selection of omic approaches and forethought about analysis techniques. We describe important considerations when designing and implementing omic-based experiments and discuss how systems biology analysis strategies can enhance efforts to obtain actionable insights in mammalian-based biologics production.

Investigation into optimal treatment intervals of facial port-wine stains using the pulsed dye laser.

BACKGROUND: Port-wine stains (PWS) affect 0.3% to 0.5% of newborns and pulsed dye laser (PDL) remains the treatment of choice. Optimal treatment intervals have not been established. OBJECTIVE: We sought to validate the optimal treatment intervals for the management of facial PWS with PDL. METHODS: In all, 24 infants with facial PWS who received at least 5 treatments with the PDL at 2-, 3-, and 4-week intervals at a private laser and skin surgery center from 2009 to 2010 were identified by a retrospective chart review. Safety and efficacy were compared by blinded investigators. RESULTS: Side effects were equivalent in all interval groups and included only expected short-term erythema, edema, purpura, and mild postinflammatory hyperpigmentation. No patient developed hypopigmentation, scarring, or infection. All interval groups showed 50% to 100% clearance of their PWS after 5 treatments. Complete or near-complete clearance was seen in 6 of 8 (75%) and 7 of 8 (87.5%) patients in the 2- and 3-week interval groups, respectively, as compared with 3 of 8 (37.5%) patients in the 4-week interval group. LIMITATIONS: This was a retrospective chart review from a single institution. Long-term side effects and recurrence rates were not assessed. CONCLUSION: We conclude that PDL treatments at 2-, 3-, and 4-week intervals are effective for the management of facial PWS in infants with minimal short-term side effects. Shorter treatment intervals may allow for relatively more rapid and more effective treatment.

Reduction of thickened flap using fractional carbon dioxide laser.

BACKGROUND AND OBJECTIVE: The paramedian forehead flap is an excellent choice when repairing a large nasal defect. However, even when carefully thinned, the flap may develop a bulky appearance, an ill-fitting contour, or trap door deformity. When on the face, these suboptimal results can be quite distressing. Surgical and non-surgical options for improvement exist. Surgical options include additional debulking and reorientation of the flap. Non-surgical options include intralesional corticosteroids or 5-flourouracil, dermabrasion, and ablative and non-ablative laser resurfacing. Each option has limited benefit as well potential side effects. STUDY DESIGN/MATERIALS AND METHODS: Case report. RESULTS: In this report, we present dramatic improvement of a thickened paramedian forehead flap using the Fraxel Re:pair, a fractional carbon dioxide (CO(2) ) laser (Solta Medical, Inc. Hayward, CA). CONCLUSION: To our knowledge, this is the first case in the literature demonstrating successful reduction of a bulky flap using a fractional ablative laser.

Long-term results after aortic valve replacement for bicuspid or tricuspid valve morphology in a Swedish population.

OBJECTIVES: Our goal was to study long-term observed and relative survival after first-time aortic valve replacement surgery with or without concomitant coronary artery bypass surgery with reference to valve morphology (i.e. bicuspid vs tricuspid). METHODS: Consecutive patients (n = 5086) from 3 Swedish hospitals, operated on between 1 January 2005 and 31 December 2016, were included. The 30-day mortality (n = 116, 2.3%) was excluded from the analysis of long-term observed and relative survival (n = 4970). Observed survival was analysed using Cox regression. Relative survival was calculated as the ratio between observed and expected survival based on data from the general Swedish population, matched for age, sex and calendar year. Risk factors for death were explored using multivariable analysis. RESULTS: During the follow-up (median 4.7 years) period, 1157 (23%) patients died. Observed survival excluding 30-day mortality was 96.6%, 82.7% and 57.6% after 1, 5 and 10 years. Compared with the general Swedish population, the relative 1-, 5- and 10-year survival rates were 99.0%, 97.5% and 89.0%. Bicuspid morphology was independently associated with higher observed and relative long-term survival. Renal dysfunction, diabetes, chronic obstructive pulmonary disease, heart failure, smoking and atrial fibrillation were associated with higher long-term mortality. Combined surgery was not associated with higher observed or relative mortality. CONCLUSIONS: Patients with a bicuspid morphology had better prognosis, matching that of the general population. With increased age, long-term relative survival compared favourably with survival in the general Swedish population. Adding coronary artery bypass surgery to an aortic valve replacement procedure did not affect long-term outcome.

Erratum: An optimized genome-wide, virus-free CRISPR screen for mammalian cells.

[This corrects the article DOI: 10.1016/j.crmeth.2021.100062.].

An optimized genome-wide, virus-free CRISPR screen for mammalian cells.

Pooled CRISPR screens have been widely applied to mammalian and other organisms to elucidate the interplay between genes and phenotypes of interest. The most popular method for delivering the CRISPR components into mammalian cells is lentivirus based. However, because lentivirus is not always an option, virus-free protocols are starting to emerge. Here, we demonstrate an improved virus-free, genome-wide CRISPR screening platform for Chinese hamster ovary cells with 75,488 gRNAs targeting 15,028 genes. Each gRNA expression cassette in the library is precisely integrated into a genomic landing pad, resulting in a very high percentage of single gRNA insertions and minimal clonal variation. Using this platform, we perform a negative selection screen on cell proliferation that identifies 1,980 genes that affect proliferation and a positive selection screen on the toxic endoplasmic reticulum stress inducer, tunicamycin, that identifies 77 gene knockouts that improve survivability.

Sensitivity and specificity for detecting basal cell carcinomas in Mohs excisions with confocal fluorescence mosaicing microscopy.

Recent studies have demonstrated the ability of confocal fluorescence mosaicing microscopy to rapidly detect basal cell carcinomas (BCCs) directly in thick and fresh Mohs surgical excisions. Mosaics of confocal images display large areas of tissue with high resolution and magnification equivalent to 2x, which is the standard magnification when examining pathology. Comparison of mosaics to Mohs frozen histopathology was shown to be excellent for all types of BCCs. However, comparisons in the previous studies were visual and qualitative. In this work, we report the results of a semiquantitative preclinical study in which 45 confocal mosaics are blindly evaluated for the presence (or absence) of BCC tumor. The evaluations are made by two clinicians: a senior Mohs surgeon with prior expertise in interpreting confocal images, and a novice Mohs fellow with limited experience. The blinded evaluation is compared to the gold standard of frozen histopathology. BCCs are detected with an overall sensitivity of 96.6%, specificity of 89.2%, positive predictive value of 93.0%, and negative predictive value of 94.7%. The results demonstrate the potential clinical utility of confocal mosaicing microscopy toward rapid surgical pathology at the bedside to expedite and guide surgery.