Cytochrome C

//  荧光蛋白作为分子探针可以特异性地标记目标蛋白、细胞结构及细胞器等，在活细胞成像和超分辨率显微技术中发挥着至关重要的作用。其亮度和光稳定性是衡量荧光蛋白在荧光显微成像中应用价值的核心指标。自从1992年成功克隆出第一个绿色荧光蛋白（GFP）以来，科学家们就致力于提升荧光蛋白的这两个关键属性，以便拓展其在成像技术中的应用范围。最近，一种源自C. uchidae水母的绿色荧光蛋白被发现具有特别强的光稳定性，并且远超已知的任何荧光蛋白，经过工程改造后，其亮度不亚于mNeonGreen，因而被命名为StayGold2。然而，StayGold本身是二聚体，限制了其作为荧光标记工具的应用范围。2024年2月26日，西湖实验室Kiryl Piatkevich课题组联合莫斯科国立大学Fedor Subach课题组在Nature Methods杂志发表了题为《Bright and stable monomeric fluorescent protein derived from StayGold》的研究论文。他们采用定向进化的策略，成功开发出了StayGold的单体版本，命名为mBaoJin。该荧光单体不仅继承了原蛋白的优异特性，还具备了更广泛的应用潜力。通过对mBaoJin在不同pH条件下的晶体结构进行分析，并将其与StayGold及其他主流荧光蛋白进行比较，他们还揭示了单体化所必须的关键氨基酸突变，并进一步阐明了其优异光稳定性的分子机制。mBaoJin的亮度高和光稳定性强，加之其出色的化学稳定性，使其成为研究细胞及亚细胞结构形态和动态变化过程中，特别是超分辨显微成像技术和膨胀显微技术的理想荧光蛋白工具。文章链接：https://www.nature.com/articles/s41592-024-02203-y论文截图图1. 单体化表达载体的示意图01单体化 StayGold为了单体化二聚体绿色荧光蛋白StayGold，作者构建图1a的蛋白表达载体，将SatyGold的突变文库与AraC蛋白的DNA结合域（AraCDNA）进行融合。当突变体是单体时，与AraCDNA融合的突变体蛋白驱动报告基因mTagBFP的表达水平比较低；当突变体是二聚体时，与AraCDNA融合的突变体蛋白驱动报告基因mTagBFP的表达水平比较高。在每轮随机突变过程中，作者挑选蓝色荧光比较暗而绿色荧光最亮的菌落，并用液相色谱确认它们的寡聚态。经过八轮定向进化后，最终确定一个亮度最高的绿色荧光蛋白单体，同时使用mNeonGreen蛋白的N端和C端作为连接肽段，以确保其在哺乳动物细胞中稳定表达，并将其名为mBaoJin。与StayGold的氨基酸序列比对，mBaoJin具有以下突变：S55T、H77R、E80G、Q140P、H141Q、C165Y、N171Y和T201A。图2. 利用液相色谱和HeLa细胞验证mBaoJin蛋白的单体状态02验证mBaoJin的单体状态为了检测mBaoJin在哺乳动物细胞中的是否为单体状态，作者在HeLa细胞中表达了与内质网锚定结构域（CytERM）融合的mBaoJin，统计没有出现涡旋结构的健康细胞的比例。同时使用了StayGold的串联和单体化版本（td8oxStayGold、StayGold-E138D和mStayGold，也称为QC2-6 FIQ）以及经过验证的单体（mEGFP、mClover3、mGreenLantern）和弱二聚体（EGFP、Venus）作为比较。mBaoJin的得分为93.7%，在细胞中达到了单体性的阈值。这些结果确定了mBaoJin是一种单体蛋白。03mBaoJin的晶体结构在文中，作者解析了mBaoJin在pH为 6.5、4.6和8.5时的X射线晶体结构，并与StayGold二聚体结构进行比对，发现mBaoJin在不同pH值下的结构差异不大，mBaoJin二聚界面之间的氨基酸相互作用比较少，mBaoJin的所有结构中荧光团附近的氯离子口袋相似且与StayGold相似。为了进一步理解mBaoJin增强光稳定性的分子基础，作者对与荧光基团相互作用的位点134、137、152（按PDB中的编号）的氨基酸进行了点突变。结果发现，mBaoJin/N137K、mBaoJin/S134P和mBaoJin/V152E突变体的光漂白速度比mBaoJin快1.5-6.5和4.5倍。比较耐光漂白的mBaoJin蛋白和较不耐光漂白的mNeonGreen及EGFP蛋白的荧光团环境，发现耐光漂白的mBaoJin蛋白在荧光基团周围形成更多的氢键和疏水键。04在细胞和纯化的蛋白中对mBaoJin的理化性质进行表征在验证mBaoJin为单体蛋白后，作者进一步对纯化的mBaoJin蛋白的光谱和生化属性进行了表征。实验发现，mBaoJin的吸收和发射光谱与StayGold相似。然而，mBaoJin的分子亮度比StayGold低1.09倍，但比mNeonGreen亮1.24倍。在低激发强度（约13 mW/mm^2，活细胞成像条件）下，mBaoJin的光稳定性比EGFP、mNeonGreen和mGreenLantern分别高15倍、9倍和130倍，尽管在高功率（约120 mW/mm^2）下这种差异不那么显著。mBaoJin在37℃下快速成熟，成熟时间半值为7.5分钟，比StayGold、mNeonGreen和EGFP分别快1.8倍、2.2倍和1.8倍，是最快成熟的荧光蛋白之一。此外，mBaoJin的荧光表现出极高的化学稳定性，与其他单体GFPs相比，mBaoJin具有较高的pH稳定性，pKa值为4.37。mBaoJin在6M GdnHCl中孵育24小时后，其荧光增加了18%，而化学稳定性最强的荧光蛋白之一hfYFP的荧光仅增加了1%，StayGold的荧光减少了2%，mNeonGreen的荧光则完全猝灭。图3. mBaoJin晶体结构、成熟时间、pKa值、细胞内亮度、膨胀显微前处理耐受性和光稳定性的表征在HEK和HeLa细胞中，通过P2A使 mBaoJin与红色荧光蛋白（FusionRed或mCherry）共表达，红色荧光则对蛋白的表达水平进行归一化。与EGFP相比，mBaoJin在HEK和HeLa细胞中的亮度一致地高出约70-140%，同时与(n1)StayGold(c4)和mStayGold相当。此外，mBaoJin在经过膨胀显微前处理的HEK细胞中，其亮度分别比mNeonGreen、hfYFP和mStayGold高出17倍、1.4倍、3.8倍。在HEK细胞进行的光漂白实验中，作者使用了比平时活细胞成像高3.5倍的照明功率（即50.5 mW/mm^2），以便在较短时间内观察到光漂白率的差异。除了StayGold及其衍生荧光蛋白比mBaoJin高1.9至2.3倍的光稳定性，mBaoJin的光漂白速度比其他现有的GFPs慢了4.6至85倍。StayGold的串联二聚体td8ox2StayGold，在亮度和光稳定性方面超过了所有测试的GFPs，在HEK细胞中表现最出色。05mBaoJin的活细胞成像和超分辨率显微成像为了验证mBaoJin能否作为荧光标签，标记目标蛋白在细胞中的分布，将其与结构蛋白融合后成像，包括vimentin，mitochondrial presequence of human cytochrome c oxidase subunit VIII，H2B，keratin，α-tubulin，β-actin等，实验结果如图4所示，mBaoJin可以很好标记结构蛋白在细胞中的位置和形状。作者使用超分辨率共聚焦显微镜对mBaoJin和mNeoGreen与α-tubulin的融合蛋白进行长达60分钟的成像，mBaoJin没有发生光漂白，而在相同条件下，mNeonGreen比初始荧光值降低了25%。接着使用稀疏去卷积结构照明显微镜对活细胞中的肌动蛋白纤维进行成像，在更高的照明功率（150-200 mW/mm2）下，mBaoJin的光稳定性比mNeonGreen高出2.5-4倍。图4. mBaoJin融合蛋白的成像和膨胀显微成像代表图06在线虫活体神经元中对表达的mNeonGreen和mBaoJin进行表征在线虫神经元中，mBaoJin的亮度比mNeonGreen高出约2倍，在连续的宽场照明下，mBaoJin展现了比mNeonGreen慢4倍的光漂白速率，在15分钟的光漂白后保留了超过50%的初始荧光。图5. mBaoJin在线虫神经元内表达的代表图及对其亮度和光稳定性的检测07在小鼠皮层神经元中对表达的绿色荧光蛋白进行表征在活的小鼠脑组织中，mBaoJin比mNeonGreen亮1.7倍，但比mStayGold暗1.7倍。然而，在PFA固定的脑组织中，mStayGold和mBaoJin表现出相当的亮度和光稳定性，显著优于mNeonGreen。由于mBaoJin的高化学稳定性，将其应用在HeLa细胞的线粒体、微丝微管以及小鼠脑组织的膨胀显微成像中。重要的是，mBaoJin在膨胀的脑组织中也保持了其高光稳定性，使得能够进行大体积的超分辨率成像，同时减少光漂白。图6. mBaoJin在小鼠大脑皮层神经元中表达的代表图及对其亮度和光稳定性的检测值得一提的是，先后有三个课题组尝试将StayGold改造成绿色荧光蛋白单体形式。Miyawaki课题组通过分子工程手段，成功开发了两个StayGold单体，mStayGold和mStayGold2（StayGold前面的m是monomeric的缩写），这两种单体在保持耐光漂白性的基础上，实现了亮度的进一步提升1。此外，Mohan K. Balasubramanian课题组联合其他两个课题组发现StayGold的一个点突变StayGold（E138D）在液相色谱实验中呈现单体状态，并且其亮度和光稳定性与StayGold相当3。Kiryl D. Piatkevich课题组联合莫斯科国立大学Fedor Subach课题组基于StayGold开发了绿色荧光蛋白单体mBaoJin，在细胞中其亮度和耐光漂白性均显著优于mNeonGreen，尽管其耐光漂白性能与mStayGold相比稍逊，但在膨胀显微成像中，其保留的荧光优于mNeonGreen和mStayGold。西湖实验室Kiryl D. Piatkevich研究员和莫斯科国立大学Fedor V. Subach教授为论文的共同通讯作者，西湖大学博士研究生张汉斌和莫斯科国立大学Gleb D. Lesnov博士研究生为本文的共同第一作者，西湖大学博士研究生张文皓和科研助理Stavrini Papadaki参与了部分研究工作。本项目获得了国家自然基金委、西湖实验室以及西湖教育基金会的资助；项目实施过程中得到了西湖大学实验动物中心和成像平台的大力支持。论文中用到的质粒可通过西湖实验室的质粒平台免费获取：https://wekwikgene.wllsb.edu.cn/ 。另外，Kiryl D. Piatkevich在2023年发表了一篇对近红外荧光蛋白进行系统性表征的论文4，相关的质粒也可在西湖实验室的质粒平台获取。WeKwikGene是中国第一个质粒库，由Piatkevich教授发起，由西湖生命科学和生物医学实验室资助。所有质粒都经过严格的质量控制和测试。在网站上，您可以找到每个质粒的完整序列，同时它提供了用户友好的体验，支持用户帐户和实验室页面的创建，并提供了增加科学工作可见性的机会。WeKwikGene保证高质量质粒的快速和免费传递，以支持和促进开放科学。实验室招聘：Kiryl D. Piatkevich博士的研究方向是研发和设计尖端分子和成像技术，用于分析、控制和修复复杂生物系统，如大脑。设计和开发用于光学记录和操控神经元活动的遗传编码分子工具，以及用于神经元解剖学追踪的荧光探针。Piatkevich实验室将系统地应用其研发的技术来揭示脑紊乱的分子机制并揭示神经编码的基本原理。这是一个跨学科研究计划，涵盖合成和化学生物学、生物工程、生物化学、生物技术、分子体内成像、神经科学、机器人和细胞生物学等领域，期待有兴趣的优秀博后加入，共同探索大脑未解之谜！招聘链接：https://www.westlake.edu.cn/Careers/OpenPositions/PostDoctoral/202112/t20211216_17044.shtml实验室网站：https://www.piatkevich-lab.com/ 参考文献：Ando R, Shimozono S, Ago H, Takagi M, Sugiyama M, Kurokawa H, et al. StayGold variants for molecular fusion and membrane-targeting applications. Nat Methods 2023.Hirano M, Ando R, Shimozono S, Sugiyama M, Takeda N, Kurokawa H, et al. A highly photostable and bright green fluorescent protein. Nat Biotechnol 2022, 40(7): 1132-1142.Ivorra-Molla E, Akhuli D, Mcandrew MBL, Scott W, Kumar L, Palani S, et al. A monomeric StayGold fluorescent protein. Nat Biotechnol 2023.Zhang H, Papadaki S, Sun X, Wang X, Drobizhev M, Yao L, et al. Quantitative assessment of near-infrared fluorescent proteins. Nat Methods 2023, 20(10): 1605-1616.文章来源 / Kiryl D. Piatkevich团队编辑 / 张玉璇校对 / 朱逸青审核 / 李凯娜

Product Sales Excluding Veklury Increased 5% Year-Over-Year to $6.4 billion Biktarvy Sales Increased 12% Year-Over-Year to $3.1 billion Oncology Sales Increased 33% Year-Over-Year to $769 million FOSTER CITY, Calif.--(BUSINESS WIRE)-- Gilead Sciences, Inc., (Nasdaq: GILD) announced today its results of operations for the third quarter of 2023. “Gilead has now delivered two years of consistent growth in our base business. In the third quarter, this continued growth was driven by both Virology and Oncology,” said Daniel O’Day, Gilead's Chairman and Chief Executive Officer. “Our clinical momentum also remains strong, and highlights this quarter included new data on Trodelvy with pembrolizumab in first-line metastatic non-small cell lung cancer. In Virology, we completed enrollment for Phase 3 trials of lenacapavir for HIV prevention and oral obeldesivir for COVID-19. We are looking forward to advancing these and other potential new options for patients over the coming months.” Third Quarter 2023 Financial Results Total third quarter 2023 revenue of $7.1 billion was flat compared to the same period in 2022, primarily driven by increased sales in Oncology and HIV, offset by lower Veklury® (remdesivir) and chronic hepatitis C virus (“HCV”) product sales. Diluted Earnings Per Share (“EPS”) increased to $1.73 for the third quarter of 2023 compared to $1.42 for the same period in 2022, and non-GAAP diluted EPS increased to $2.29 for the third quarter of 2023 compared to $1.90 for the same period in 2022. The increases in GAAP and non-GAAP diluted EPS were primarily driven by lower tax expense, partially offset by net higher total costs and expenses. As of September 30, 2023, Gilead had $8.0 billion of cash, cash equivalents and marketable debt securities, up from $7.6 billion as of December 31, 2022. During the third quarter of 2023, Gilead generated $1.8 billion in operating cash flow. During the third quarter of 2023, Gilead repaid $2.3 billion of debt, paid dividends of $953 million and repurchased $300 million of common stock. Additionally, Gilead issued senior unsecured notes in an aggregate principal amount of $2.0 billion. Product Sales Performance Total third quarter 2023 product sales of $7.0 billion was flat compared to the same period in 2022, reflecting continued growth in the base business, offset by lower Veklury sales. Total product sales, excluding Veklury, increased 5% to $6.4 billion in the third quarter of 2023 compared to the same period in 2022, primarily due to increased sales in Oncology and HIV, partially offset by lower HCV sales. HIV product sales increased 4% to $4.7 billion in the third quarter of 2023 compared to the same period in 2022, primarily driven by higher demand and channel inventory dynamics, partially offset by lower average realized price due to a shift in channel mix. Biktarvy® (bictegravir 50mg/emtricitabine 200mg (“FTC”)/tenofovir alafenamide 25mg (“TAF”)) sales increased 12% year-over-year in the third quarter of 2023, primarily driven by higher demand, as well as favorable channel inventory. Descovy® (FTC 200mg/TAF 25mg) sales increased 2% year-over-year in the third quarter of 2023, primarily driven by higher demand, partially offset by pricing dynamics in the United States. The Liver Disease portfolio sales, which includes HCV, chronic hepatitis B virus (“HBV”), and chronic hepatitis delta virus (“HDV”), decreased 10% to $706 million in the third quarter of 2023 compared to the same period in 2022, as higher HCV patient starts were more than offset by unfavorable pricing dynamics, primarily due to the resolution of a rebate claim in HCV in the third quarter of 2022. Cell Therapy product sales increased 22% to $486 million in the third quarter of 2023 compared to the same period in 2022. Yescarta® (axicabtagene ciloleucel) sales increased 23% year-over-year to $391 million in the third quarter of 2023, primarily driven by strong demand in the second- and third-line settings for relapsed or refractory (“R/R”) large B-cell lymphoma (“LBCL”) outside of the United States. Tecartus® (brexucabtagene autoleucel) sales increased 18% year-over-year to $96 million in the third quarter of 2023, primarily driven by increased demand in R/R mantle cell lymphoma (“MCL”) and R/R adult acute lymphoblastic leukemia (“ALL”). Trodelvy® (sacituzumab govitecan-hziy) sales increased 58% to $283 million in the third quarter of 2023 compared to the same period in 2022, primarily driven by higher demand in both the United States and Europe. Veklury sales decreased 31% to $636 million for the third quarter of 2023 compared to the same period in 2022, primarily driven by lower rates of COVID-19 related hospitalizations in all regions. Veklury sales generally reflect COVID-19 related rates and severity of infections and hospitalizations, as well as the availability, uptake and effectiveness of vaccinations and alternative treatments for COVID-19. Third Quarter 2023 Product Gross Margin, Operating Expenses and Effective Tax Rate Product gross margin was 77.6% for the third quarter of 2023 compared to 80.0% for the same period in 2022, primarily driven by intangible asset amortization expenses related to the pretreated HR+/HER2- metastatic breast cancer indication for Trodelvy following its approval in February 2023, as well as product mix. Non-GAAP product gross margin was 85.9% for the third quarter of 2023 compared to 86.8% in the same period in 2022, primarily driven by product mix. Research & development (“R&D”) expenses for the third quarter of 2023 were $1.5 billion compared to $1.1 billion in the same period in 2022. Non-GAAP R&D expenses for the third quarter of 2023 were $1.5 billion compared to $1.2 billion in the same period in 2022. The increases in GAAP and non-GAAP R&D expenses were primarily driven by increased clinical activity, as well as costs associated with the discontinuation of two Phase 3 magrolimab studies. Acquired in-process research and development (“IPR&D”) expenses for the third quarter of 2023 were $91 million, driven by an upfront payment related to the Tentarix Biotherapeutics Inc. (“Tentarix”) collaboration and other collaboration-related activities. Selling, general and administrative (“SG&A”) expenses and non-GAAP SG&A expenses for the third quarter of 2023 were $1.3 billion compared to $1.2 billion in the same period in 2022. The increases in GAAP and non-GAAP SG&A expenses were primarily driven by increased commercial activities in Oncology. The effective tax rate (“ETR”) for the third quarter of 2023 was 6.3% compared to 26.6% for the same period in 2022, and non-GAAP ETR for the third quarter of 2023 was 7.0% compared to 22.4% for the same period in 2022. Both ETR and non-GAAP ETR decreases were primarily driven by a decrease in tax reserves as a result of reaching agreement with a tax authority on certain tax positions. Guidance and Outlook For the full-year, Gilead expects: Total product sales between $26.7 billion and $26.9 billion, compared to $26.3 billion and $26.7 billion previously. Total product sales, excluding Veklury, between $24.8 billion and $25.0 billion, compared to $24.6 billion and $25.0 billion previously. Total Veklury sales of approximately $1.9 billion, compared to approximately $1.7 billion previously. Diluted earnings per share between $4.55 and $4.75, compared to $4.50 and $4.85 previously. Non-GAAP diluted earnings per share between $6.65 and $6.85, compared to $6.45 and $6.80 previously. Additional information and a reconciliation between GAAP and non-GAAP financial information for the 2023 guidance is provided in the accompanying tables. Also see the Forward-Looking Statements described below. The financial guidance is subject to a number of risks and uncertainties, including uncertainty around the duration and magnitude of the COVID-19 pandemic. Key Updates Since Our Last Quarterly Release Virology Received U.S. Food and Drug Administration (“FDA”) and European Commission approval to extend the use of Veklury to treat COVID-19 in appropriate patients with mild to severe hepatic impairment. Presented new data at the European AIDS Conference 2023, including three-year outcomes from BICSTaR, an ongoing real-world study evaluating Biktarvy in people with HIV who have a high burden of co-morbidities, and multiple analyses from the Phase 2/3 CAPELLA study of lenacapavir in people with multi-drug resistant HIV. Announced PURPOSE 5, a Phase 2 clinical trial to assess the persistence of lenacapavir compared with FTC/tenofovir disoproxil fumarate in people who may benefit from PrEP in Europe. The use of lenacapavir for PrEP is investigational. Presented new data at Infectious Disease Week 2023 demonstrating Biktarvy’s efficacy and safety pro a broad range of people with HIV as well as two-year outcomes from the CAPELLA study of lenacapavir. Additionally, presented data on the safety pro Veklury across vulnerable populations and the drug-drug interaction pro the investigational oral antiviral obeldesivir, as well as new in vitro data on antiviral activity of Veklury and obeldesivir against recent SARS-CoV-2 Omicron subvariants. Discontinued the Phase 3 BIRCH trial of obeldesivir in non-hospitalized participants who are at high risk for developing severe COVID-19. The decision was based on lower-than-expected COVID-19 incidence rates and related hospitalizations or all-cause death by Day 29, and does not reflect any safety or efficacy concerns. Announced the Phase 3 OAKTREE trial of obeldesivir in non-hospitalized participants without risk factors for developing severe COVID-19 is approaching full enrollment, and remains unaffected by the BIRCH decision. Announced plans to present at the American Association for the Study of Liver Diseases 2023 annual meeting. Presentations will include new and long-term data across Gilead’s Liver Disease portfolio. Announced a collaboration with Assembly Biosciences, Inc. (“Assembly”) to advance the research and development of novel antiviral therapies, including in herpesviruses, HBV and HDV. Oncology Presented promising early data at the 2023 World Conference on Lung Cancer from the Phase 2 EVOKE-02 study evaluating the investigational use of Trodelvy in combination with Keytruda® (pembrolizumab) as a first-line treatment in patients with advanced or metastatic non-small cell lung cancer without actionable genomic alterations. The study demonstrated clinical activity in patients with PD-L1 tumor proportion score (“TPS”) >50% in Cohort A and PD-L1 TPS<50% in Cohort B, including early and durable responses, as well as safety consistent with the known safety pro each agent. Presented data at the European Society of Medical Oncology 2023 meeting from the Phase 2 TROPiCS-03 basket study demonstrating encouraging activity of Trodelvy for investigational use in both advanced head and neck squamous cell carcinoma and extensive stage small cell lung cancer cohorts, as well as other data in breast and urothelial cancer. Additionally, presented a comparative analysis, adjusting for trial differences, on Yescarta relative to bispecific antibodies in 3L+ R/R LBCL. Announced abstracts for the American Society of Hematology 2023 Annual Meeting, including updated Phase 1 data evaluating the Arcellx, Inc. (“Arcellx”) partnered CART-ddBCMA in R/R multiple myeloma, demonstrating continued deep and durable responses with 22 months follow-up. Additionally announced abstracts for long-term follow-up across Yescarta trials in R/R LBCL, first-line high-risk LBCL, and R/R follicular lymphoma, as well as real-world data for Tecartus in R/R MCL and B-cell ALL. Announced results from the Phase 2 ALYCANTE study, led and sponsored by the French collaborative group LYSA/LYSARC, evaluating Yescarta in transplant-ineligible patients with second-line R/R LBCL, which demonstrated high response rates and durable remission. Presented initial data, alongside Arcus Biosciences, Inc. (“Arcus”) at the American Society of Clinical Oncology Monthly Plenary Session from the Phase 2 EDGE-Gastric study of domvanalimab in combination with zimberelimab and chemotherapy in first-line locally advanced unresectable or metastatic gastric, gastroesophageal junction, or esophageal adenocarcinoma. The results showed encouraging overall response rates and six-month landmark progression-free survival rate. Discontinued the Phase 3 ENHANCE-2 study of magrolimab in first-line acute myeloid leukemia (“AML”) with TP53 mutations based on an ad hoc analysis and following review by an independent data monitoring committee. Additionally announced that the U.S. FDA placed a partial clinical hold on magrolimab studies in AML that paused enrollment, though previously enrolled patients may continue to receive the study medicine. Announced a collaboration with Tentarix to discover and develop novel therapies across oncology and inflammation, using Tentarix’s proprietary Tentacles platform. Announced a collaboration with Epicrispr Biotechnologies, Inc. (“Epic Bio”) to develop next-generation cancer cell therapies using Epic Bio’s proprietary gene regulation platform. Corporate Issued $2.0 billion aggregate principal amount of senior unsecured notes in a registered offering, comprised of $1.0 billion principal amount of 5.25% senior notes due in 2033 and $1.0 billion principal amount of 5.55% senior notes due in 2053, and repaid debt of $2.3 billion. The company’s Board of Directors declared a quarterly dividend of $0.75 per share of common stock for the fourth quarter of 2023. The dividend is payable on December 28, 2023, to stockholders of record at the close of business on December 15, 2023. Future dividends will be subject to Board approval. Certain amounts and percentages in this press release may not sum or recalculate due to rounding. Conference Call At 1:30 p.m. Pacific Time today, Gilead will host a conference call to discuss Gilead’s results. A live webcast will be available on and will be archived on for one year. Non-GAAP Financial Information The information presented in this document has been prepared in accordance with U.S. generally accepted accounting principles (“GAAP”), unless otherwise noted as non-GAAP. Management believes non-GAAP information is useful for investors, when considered in conjunction with Gilead’s GAAP financial information, because management uses such information internally for its operating, budgeting and financial planning purposes. Non-GAAP information is not prepared under a comprehensive set of accounting rules and should only be used to supplement an understanding of Gilead’s operating results as reported under GAAP. Non-GAAP financial information generally excludes acquisition-related expenses including amortization of acquired intangible assets and inventory step-up charges, and other items that are considered unusual or not representative of underlying trends of Gilead’s business, fair value adjustments of equity securities and discrete and related tax charges or benefits associated with changes in tax related laws and guidelines. Although Gilead consistently excludes the amortization of acquired intangible assets from the non-GAAP financial information, management believes that it is important for investors to understand that such intangible assets were recorded as part of acquisitions and contribute to ongoing revenue generation. Non-GAAP measures may be defined and calculated differently by other companies in the same industry. Reconciliations of the non-GAAP financial measures to the most directly comparable GAAP financial measures are provided in the accompanying tables. About Gilead Sciences Gilead Sciences, Inc. is a biopharmaceutical company that has pursued and achieved breakthroughs in medicine for more than three decades, with the goal of creating a healthier world for all people. The company is committed to advancing innovative medicines to prevent and treat life-threatening diseases, including HIV, viral hepatitis, COVID-19 and cancer. Gilead operates in more than 35 countries worldwide, with headquarters in Foster City, California. Forward-Looking Statements Statements included in this press release that are not historical in nature are forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. Gilead cautions readers that forward-looking statements are subject to certain risks and uncertainties that could cause actual results to differ materially. These risks and uncertainties include those relating to: Gilead’s ability to achieve its anticipated full year 2023 financial results, including as a result of the uncertainty of the amount and timing of Veklury revenues; Gilead’s ability to make progress on any of its long-term ambitions or strategic priorities laid out in its corporate strategy; Gilead’s ability to accelerate or sustain revenues for its virology, oncology and other programs; Gilead’s ability to realize the potential benefits of acquisitions, collaborations or licensing arrangements, including the arrangements with Arcus, Arcellx, Assembly, Epic Bio, and Tentarix; patent protection and estimated loss of exclusivity for our products and product candidates; Gilead’s ability to initiate, progress or complete clinical trials within currently anticipated timeframes or at all, the possibility of unfavorable results from ongoing and additional clinical trials, including those involving Biktarvy, Tecartus, Trodelvy, Veklury, Yescarta, CART-ddBCMA, domvanalimab, lenacapavir, magrolimab, obeldesivir, and zimberelimab, and the risk that safety and efficacy data from clinical trials may not warrant further development of Gilead’s product candidates or the product candidates of Gilead’s strategic partners; Gilead’s ability to submit new drug applications for new product candidates or expanded indications in the currently anticipated timelines; Gilead’s ability to receive regulatory approvals in a timely manner or at all, and the risk that any such approvals, if granted, may be subject to significant limitations on use; Gilead’s ability to successfully commercialize its products; the risk of potential disruptions to the manufacturing and supply chain of Gilead’s products; pricing and reimbursement pressures from government agencies and other third parties, including required rebates and other discounts; a larger than anticipated shift in payer mix to more highly discounted payer segments; market share and price erosion caused by the introduction of generic versions of Gilead products; the risk that physicians and patients may not see advantages of these products over other therapies and may therefore be reluctant to prescribe the products, including Veklury; and other risks identified from time to time in Gilead’s reports filed with the SEC, including annual reports on Form 10-K, quarterly reports on Form 10-Q and current reports on Form 8-K. In addition, Gilead makes estimates and judgments that affect the reported amounts of assets, liabilities, revenues and expenses and related disclosures. Gilead bases its estimates on historical experience and on various other market specific and other relevant assumptions that it believes to be reasonable under the circumstances, the results of which form the basis for making judgments about the carrying values of assets and liabilities that are not readily apparent from other sources. There may be other factors of which Gilead is not currently aware that may affect matters discussed in the forward-looking statements and may also cause actual results to differ significantly from these estimates. Further, results for the quarter ended September 30, 2023 are not necessarily indicative of operating results for any future periods. Gilead directs readers to its press releases, annual reports on Form 10-K, quarterly reports on Form 10-Q and other subsequent disclosure documents filed with the SEC. Gilead claims the protection of the Safe Harbor contained in the Private Securities Litigation Reform Act of 1995 for forward-looking statements. The reader is cautioned that forward-looking statements are not guarantees of future performance and is cautioned not to place undue reliance on these forward-looking statements. All forward-looking statements are based on information currently available to Gilead and Gilead assumes no obligation to update or supplement any such forward-looking statements other than as required by law. Any forward-looking statements speak only as of the date hereof or as of the dates indicated in the statements. Gilead owns or has rights to various trademarks, copyrights and trade names used in its business, including the following: GILEAD®, GILEAD SCIENCES®, KITE™, AMBISOME®, ATRIPLA®, BIKTARVY®, CAYSTON®, COMPLERA®, DESCOVY®, DESCOVY FOR PREP®, EMTRIVA®, EPCLUSA®, EVIPLERA®, GENVOYA®, HARVONI®, HEPCLUDEX®, HEPSERA®, JYSELECA®, LETAIRIS®, ODEFSEY®, RANEXA®, SOVALDI®, STRIBILD®, SUNLENCA® , TECARTUS®, TRODELVY®, TRUVADA®, TRUVADA FOR PREP®, TYBOST®, VEKLURY®, VEMLIDY®, VIREAD®, VOSEVI®, YESCARTA® and ZYDELIG®. KEYTRUDA® is a registered trademark of Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, United States. Other trademarks are the property of their respective owners. For more information on Gilead Sciences, Inc., please visit or call the Gilead Public Affairs Department at 1-800-GILEAD-5 (1-800-445-3235). GILEAD SCIENCES, INC. CONDENSED CONSOLIDATED STATEMENTS OF INCOME (unaudited) Three Months Ended Nine Months Ended September 30, September 30, (in millions, except per share amounts) 2023 2022 2023 2022 Revenues: Product sales $ 6,994 $ 6,978 $ 19,864 $ 19,650 Royalty, contract and other revenues 56 64 138 242 Total revenues 7,051 7,042 20,002 19,892 Costs and expenses: Cost of goods sold 1,565 1,395 4,408 4,261 Research and development expenses 1,457 1,149 4,310 3,429 Acquired in-process research and development expenses 91 448 808 786 In-process research and development impairment — — — 2,700 Selling, general and administrative expenses 1,315 1,213 4,482 3,653 Total costs and expenses 4,428 4,205 14,009 14,829 Operating income 2,623 2,837 5,993 5,063 Interest expense (232 ) (229 ) (692 ) (709 ) Other income (expense), net (72 ) (176 ) (95 ) (571 ) Income before income taxes 2,318 2,432 5,206 3,783 Income tax expense (146 ) (646 ) (1,010 ) (850 ) Net income 2,172 1,786 4,196 2,933 Net loss attributable to noncontrolling interest 8 3 40 19 Net income attributable to Gilead $ 2,180 $ 1,789 $ 4,236 $ 2,952 Basic earnings per share attributable to Gilead $ 1.75 $ 1.43 $ 3.39 $ 2.35 Shares used in basic earnings per share attributable to Gilead calculation 1,248 1,255 1,249 1,255 Diluted earnings per share attributable to Gilead $ 1.73 $ 1.42 $ 3.37 $ 2.34 Shares used in diluted earnings per share attributable to Gilead calculation 1,257 1,261 1,259 1,261 Cash dividends declared per share $ 0.75 $ 0.73 $ 2.25 $ 2.19 Research and development expenses as a % of revenues 20.7 % 16.3 % 21.5 % 17.2 % Selling, general and administrative expenses as a % of revenues 18.6 % 17.2 % 22.4 % 18.4 % GILEAD SCIENCES, INC. TOTAL REVENUE SUMMARY (unaudited) Three Months Ended Nine Months Ended September 30, September 30, (in millions, except percentages) 2023 2022 Change 2023 2022 Change Product sales: HIV $ 4,667 $ 4,487 4 % $ 13,482 $ 12,422 9 % Oncology 769 578 33 % 2,167 1,525 42 % Liver Disease 706 788 (10 )% 2,093 2,104 (1 )% Other 216 200 8 % 658 693 (5 )% Total product sales excluding Veklury 6,358 6,053 5 % 18,400 16,745 10 % Veklury 636 925 (31 )% 1,465 2,905 (50 )% Total product sales 6,994 6,978 — % 19,864 19,650 1 % Royalty, contract and other revenues 56 64 (13 )% 138 242 (43 )% Total revenues $ 7,051 $ 7,042 — % $ 20,002 $ 19,892 1 % GILEAD SCIENCES, INC. NON-GAAP FINANCIAL INFORMATION(1) (unaudited) Three Months Ended Nine Months Ended September 30, September 30, (in millions, except percentages) 2023 2022 Change 2023 2022 Change Non-GAAP: Cost of goods sold $ 985 $ 923 7% $ 2,717 $ 2,634 3% Research and development expenses $ 1,453 $ 1,173 24% $ 4,268 $ 3,425 25% Acquired IPR&D expenses $ 91 $ 448 (80)% $ 808 $ 786 3% Selling, general and administrative expenses $ 1,298 $ 1,212 7% $ 4,464 $ 3,566 25% Other income (expense), net $ 96 $ 20 NM $ 261 $ 25 NM Diluted EPS $ 2.29 $ 1.90 21% $ 5.00 $ 5.59 (11)% Product gross margin 85.9 % 86.8 % -85 bps 86.3 % 86.6 % -27 bps Research and development expenses as a % of revenues 20.6 % 16.7 % 394 bps 21.3 % 17.2 % 412 bps Selling, general and administrative expenses as a % of revenues 18.4 % 17.2 % 120 bps 22.3 % 17.9 % 439 bps Operating margin 45.7 % 46.7 % -92 bps 38.7 % 47.7 % -894 bps Effective tax rate 7.0 % 22.4 % -1540 bps 14.5 % 20.1 % -555 bps NM - Not Meaningful (1) Refer to Non-GAAP Financial Information section above for further disclosures on non-GAAP financial measures. A reconciliation between GAAP and non-GAAP financial information is provided in the tables below. GILEAD SCIENCES, INC. RECONCILIATION OF GAAP TO NON-GAAP FINANCIAL INFORMATION (unaudited) Three Months Ended Nine Months Ended September 30, September 30, (in millions, except percentages and per share amounts) 2023 2022 2023 2022 Cost of goods sold reconciliation: GAAP cost of goods sold $ 1,565 $ 1,395 $ 4,408 $ 4,261 Acquisition-related – amortization(1) (581 ) (472 ) (1,691 ) (1,585 ) Other(2) — — — (42 ) Non-GAAP cost of goods sold $ 985 $ 923 $ 2,717 $ 2,634 Product gross margin reconciliation: GAAP product gross margin 77.6 % 80.0 % 77.8 % 78.3 % Acquisition-related – amortization(1) 8.3 % 6.8 % 8.5 % 8.1 % Other(2) — % — % — % 0.2 % Non-GAAP product gross margin 85.9 % 86.8 % 86.3 % 86.6 % Research and development expenses reconciliation: GAAP research and development expenses $ 1,457 $ 1,149 $ 4,310 $ 3,429 Acquisition-related – other costs(3) 1 24 (37 ) 13 Other(2) (5 ) — (5 ) (18 ) Non-GAAP research and development expenses $ 1,453 $ 1,173 $ 4,268 $ 3,425 IPR&D impairment reconciliation: GAAP IPR&D impairment $ — $ — $ — $ 2,700 IPR&D impairment — — — (2,700 ) Non-GAAP IPR&D impairment $ — $ — $ — $ — Selling, general and administrative expenses reconciliation: GAAP selling, general and administrative expenses $ 1,315 $ 1,213 $ 4,482 $ 3,653 Acquisition-related – other costs(3) — (2 ) (2 ) (2 ) Other(2) (17 ) 1 (17 ) (84 ) Non-GAAP selling, general and administrative expenses $ 1,298 $ 1,212 $ 4,464 $ 3,566 Operating income reconciliation: GAAP operating income $ 2,623 $ 2,836 $ 5,993 $ 5,063 Acquisition-related – amortization(1) 581 472 1,691 1,585 Acquisition-related – other costs(3) (1 ) (22 ) 39 (11 ) IPR&D impairment — — — 2,700 Other(2) 22 (1 ) 22 144 Non-GAAP operating income $ 3,224 $ 3,286 $ 7,745 $ 9,480 Operating margin reconciliation: GAAP operating margin 37.2 % 40.3 % 30.0 % 25.5 % Acquisition-related – amortization(1) 8.2 % 6.7 % 8.5 % 8.0 % Acquisition-related – other costs(3) — % (0.3 )% 0.2 % (0.1 )% IPR&D impairment — % — % — % 13.6 % Other(2) 0.3 % — % 0.1 % 0.7 % Non-GAAP operating margin 45.7 % 46.7 % 38.7 % 47.7 % Other income (expense), net reconciliation: GAAP other income (expense), net $ (72 ) $ (176 ) $ (95 ) $ (571 ) Loss from equity securities, net 168 197 356 596 Non-GAAP other income (expense), net $ 96 $ 20 $ 261 $ 25 GILEAD SCIENCES, INC. RECONCILIATION OF GAAP TO NON-GAAP FINANCIAL INFORMATION - (Continued) (unaudited) Three Months Ended Nine Months Ended September 30, September 30, (in millions, except percentages and per share amounts) 2023 2022 2023 2022 Effective tax rate reconciliation: GAAP effective tax rate 6.3 % 26.6 % 19.4 % 22.5 % Income tax effect of above non-GAAP adjustments and discrete and related tax adjustments(4) 0.7 % (4.1 )% (4.9 )% (2.4 )% Non-GAAP effective tax rate 7.0 % 22.4 % 14.5 % 20.1 % Net income attributable to Gilead reconciliation: GAAP net income attributable to Gilead $ 2,180 $ 1,789 $ 4,236 $ 2,952 Acquisition-related – amortization(1) 461 379 1,345 1,264 Acquisition-related – other costs(3) (1 ) (23 ) 31 (13 ) IPR&D impairment — — — 2,057 Loss from equity securities, net 164 198 351 570 Discrete and related tax charges(4) 58 49 314 118 Other(2) 17 — 17 104 Non-GAAP net income attributable to Gilead $ 2,879 $ 2,391 $ 6,293 $ 7,052 Diluted earnings per share reconciliation: GAAP diluted earnings per share $ 1.73 $ 1.42 $ 3.37 $ 2.34 Acquisition-related – amortization(1) 0.37 0.30 1.07 1.00 Acquisition-related – other costs(3) — (0.02 ) 0.02 (0.01 ) IPR&D impairment — — — 1.63 Loss from equity securities, net 0.13 0.16 0.28 0.45 Discrete and related tax charges(4) 0.05 0.04 0.25 0.09 Other(2) 0.01 — 0.01 0.08 Non-GAAP diluted earnings per share $ 2.29 $ 1.90 $ 5.00 $ 5.59 Non-GAAP adjustment summary: Cost of goods sold adjustments $ 581 $ 472 $ 1,691 $ 1,627 Research and development expenses adjustments 4 (24 ) 42 5 IPR&D impairment adjustments — — — 2,700 Selling, general and administrative expenses adjustments 17 1 19 86 Total non-GAAP adjustments to costs and expenses 602 450 1,752 4,418 Other income (expense), net, adjustments 168 197 356 596 Total non-GAAP adjustments before income taxes 770 646 2,108 5,014 Income tax effect of non-GAAP adjustments above (129 ) (93 ) (364 ) (1,032 ) Discrete and related tax charges(4) 58 49 314 118 Total non-GAAP adjustments after tax $ 699 $ 602 $ 2,057 $ 4,100 (1) Relates to amortization of acquired intangibles and inventory step-up charges. (2) Adjustments to Cost of goods sold and Research and development expenses primarily include various restructuring expenses during the first quarter of 2022 and third quarter of 2023. Adjustments to Selling, general and administrative expenses include donations to the Gilead Foundation, a California nonprofit organization, during the second quarter of 2022 and various restructuring expenses during the third quarter of 2023. (3) Adjustments include employee-related expenses, contingent consideration fair value adjustments and other expenses associated with Gilead’s acquisitions of MYR GmbH, MiroBio, Ltd., Tmunity Therapeutics, Inc. and XinThera, Inc. (4) Represents discrete and related deferred tax charges or benefits primarily associated with acquired intangible assets and transfers of intangible assets from a foreign subsidiary to Ireland and the United States. GILEAD SCIENCES, INC. RECONCILIATION OF GAAP TO NON-GAAP 2023 FULL-YEAR GUIDANCE(1) (unaudited) (in millions, except percentages and per share amounts) Provided February 2, 2023 Updated April 27, 2023 Updated August 3, 2023 Updated November 7, 2023 Projected product gross margin GAAP to non-GAAP reconciliation: GAAP projected product gross margin 79.0% 77.0% 77.0% 77.0% Acquisition-related expenses and other ~ 7% ~ 9% ~ 9% ~ 9% Non-GAAP projected product gross margin 86.0% 86.0% 86.0% 86.0% Projected operating income GAAP to non-GAAP reconciliation: GAAP projected operating income $9,200 - $9,800 $8,600 - $9,200 $8,000 - $8,500 $8,100 - $8,400 Acquisition-related expenses and other ~ 1,800 ~ 2,400 ~ 2,400 ~ 2,400 Non-GAAP projected operating income $11,000 - $11,600 $11,000 - $11,600 $10,400 - $10,900 $10,500 - $10,800 Projected effective tax rate GAAP to non-GAAP reconciliation: GAAP projected effective tax rate ~ 22% ~ 22% ~ 21% ~ 20% Discrete and related tax adjustments, and income tax effect of adjustments above and fair value adjustments of equity securities (~ 2%) (~ 2%) (~ 4%) (~ 4%) Non-GAAP projected effective tax rate ~ 20% ~ 20% ~ 17% ~ 16% Projected diluted EPS GAAP to non-GAAP reconciliation: GAAP projected diluted EPS $5.30 - $5.70 $4.75 - $5.15 $4.50 - $4.85 $4.55 - $4.75 Acquisition-related expenses, fair value adjustments of equity securities, discrete and related tax adjustments and other ~ 1.30 ~ 1.85 ~ 1.95 ~ 2.10 Non-GAAP projected diluted EPS $6.60 - $7.00 $6.60 - $7.00 $6.45 - $6.80 $6.65 - $6.85 (1) Our full-year guidance excludes the potential impact of any (i) acquisitions or business development transactions that have not been executed, (ii) future fair value adjustments of equity securities and (iii) discrete tax charges or benefits associated with changes in tax related laws and guidelines that have not been enacted, as Gilead is unable to project such amounts. The non-GAAP full-year guidance includes non-GAAP adjustments to actual current period results as well as adjustments for the known future impact associated with events that have already occurred, such as future amortization of our intangible assets and the future impact of discrete and related deferred tax charges or benefits primarily associated with acquired intangible assets and transfers of intangible assets from a foreign subsidiary to Ireland and the United States. GILEAD SCIENCES, INC. CONDENSED CONSOLIDATED BALANCE SHEETS (unaudited) September 30, December 31, (in millions) 2023 2022 Assets Cash, cash equivalents and marketable debt securities $ 8,021 $ 7,630 Accounts receivable, net 4,790 4,777 Inventories 3,202 2,820 Property, plant and equipment, net 5,572 5,475 Intangible assets, net 27,152 28,894 Goodwill 8,314 8,314 Other assets 5,323 5,262 Total assets $ 62,373 $ 63,171 Liabilities and Stockholders’ Equity Current liabilities $ 11,945 $ 11,237 Long-term liabilities 28,186 30,725 Stockholders’ equity(1) 22,242 21,209 Total liabilities and stockholders’ equity $ 62,373 $ 63,171 (1) As of September 30, 2023 and December 31, 2022, there were 1,247 shares of common stock issued and outstanding. GILEAD SCIENCES, INC. SELECTED CASH FLOW INFORMATION (unaudited) Three Months Ended Nine Months Ended September 30, September 30, (in millions) 2023 2022 2023 2022 Net cash provided by operating activities $ 1,756 $ 2,863 $ 5,837 $ 6,505 Net cash used in investing activities (229 ) (713 ) (1,538 ) (2,091 ) Net cash used in financing activities (1,518 ) (2,118 ) (4,026 ) (4,915 ) Effect of exchange rate changes on cash and cash equivalents (7 ) (72 ) 20 (138 ) Net change in cash and cash equivalents 1 (40 ) 293 (639 ) Cash and cash equivalents at beginning of period 5,704 4,739 5,412 5,338 Cash and cash equivalents at end of period $ 5,705 $ 4,699 $ 5,705 $ 4,699 Three Months Ended Nine Months Ended September 30, September 30, (in millions) 2023 2022 2023 2022 Net cash provided by operating activities $ 1,756 $ 2,863 $ 5,837 $ 6,505 Capital expenditures (122 ) (157 ) (370 ) (547 ) Free cash flow(1) $ 1,633 $ 2,706 $ 5,467 $ 5,958 (1) Free cash flow is a non-GAAP liquidity measure. Please refer to our disclosures in the Non-GAAP Financial Information section above. GILEAD SCIENCES, INC. PRODUCT SALES SUMMARY (unaudited) Three Months Ended Nine Months Ended September 30, September 30, (in millions) 2023 2022 2023 2022 HIV Biktarvy – U.S. $ 2,504 $ 2,286 $ 7,104 $ 6,088 Biktarvy – Europe 313 278 920 807 Biktarvy – Other International 268 201 717 577 3,085 2,766 8,741 7,472 Complera / Eviplera – U.S. 13 20 40 56 Complera / Eviplera – Europe 18 21 55 76 Complera / Eviplera – Other International 3 3 9 10 34 43 104 142 Descovy – U.S. 460 444 1,314 1,152 Descovy – Europe 25 28 75 92 Descovy – Other International 26 28 86 91 511 500 1,475 1,335 Genvoya – U.S. 433 502 1,305 1,441 Genvoya – Europe 47 71 157 220 Genvoya – Other International 23 27 81 103 503 600 1,544 1,764 Odefsey – U.S. 257 276 754 763 Odefsey – Europe 74 86 223 278 Odefsey – Other International 11 12 33 36 343 374 1,011 1,077 Stribild – U.S. 18 22 57 68 Stribild – Europe 5 7 16 23 Stribild – Other International 2 3 6 7 25 32 79 98 Truvada – U.S. 15 24 71 77 Truvada – Europe 3 3 10 12 Truvada – Other International 4 2 16 13 22 30 97 102 Revenue share – Symtuza(1) – U.S. 96 85 278 251 Revenue share – Symtuza(1) – Europe 32 40 101 126 Revenue share – Symtuza(1) – Other International 3 4 10 10 131 130 390 388 Other HIV(2) – U.S. 10 1 24 11 Other HIV(2) – Europe 3 6 11 20 Other HIV(2) – Other International — 5 6 15 13 12 41 45 Total HIV – U.S. 3,807 3,661 10,949 9,906 Total HIV – Europe 519 541 1,568 1,653 Total HIV – Other International 341 285 965 863 $ 4,667 $ 4,487 $ 13,482 $ 12,422 GILEAD SCIENCES, INC. PRODUCT SALES SUMMARY - (Continued) (unaudited) Three Months Ended Nine Months Ended September 30, September 30, (in millions) 2023 2022 2023 2022 Oncology Cell Therapy Tecartus – U.S. $ 64 $ 60 $ 179 $ 160 Tecartus – Europe 27 20 83 56 Tecartus – Other International 4 1 11 2 96 81 272 217 Yescarta – U.S. 197 210 624 528 Yescarta – Europe 154 91 408 253 Yescarta – Other International 40 16 99 42 391 317 1,130 823 Total Cell Therapy – U.S. 261 270 802 688 Total Cell Therapy – Europe 181 111 491 308 Total Cell Therapy – Other International 45 17 109 44 486 398 1,402 1,040 Trodelvy Trodelvy – U.S. 201 139 551 379 Trodelvy – Europe 62 38 169 98 Trodelvy – Other International 21 3 44 8 283 180 764 485 Total Oncology – U.S. 462 409 1,354 1,067 Total Oncology – Europe 243 149 660 407 Total Oncology – Other International 65 20 153 52 769 578 2,167 1,525 Liver Disease HCV Ledipasvir / Sofosbuvir(3) – U.S. 17 8 29 27 Ledipasvir / Sofosbuvir(3) – Europe 2 5 11 13 Ledipasvir / Sofosbuvir(3) – Other International 4 12 14 43 23 25 54 83 Sofosbuvir / Velpatasvir(4) – U.S. 215 241 643 629 Sofosbuvir / Velpatasvir(4) – Europe 76 131 250 288 Sofosbuvir / Velpatasvir(4) – Other International 85 84 266 244 377 455 1,159 1,161 Other HCV(5) – U.S. 28 34 80 88 Other HCV(5) – Europe 7 7 33 31 Other HCV(5) – Other International 3 2 10 7 38 44 123 127 Total HCV – U.S. 260 283 751 745 Total HCV – Europe 85 143 294 332 Total HCV – Other International 93 98 289 294 $ 438 $ 524 $ 1,335 $ 1,371 GILEAD SCIENCES, INC. PRODUCT SALES SUMMARY - (Continued) (unaudited) Three Months Ended Nine Months Ended September 30, September 30, (in millions) 2023 2022 2023 2022 HBV/HDV Vemlidy – U.S. $ 112 $ 129 $ 295 $ 306 Vemlidy – Europe 9 9 28 27 Vemlidy – Other International 106 90 322 289 228 228 645 622 Viread – U.S. 4 2 4 4 Viread – Europe 5 5 17 17 Viread – Other International 12 15 40 48 21 22 62 69 Other HBV/HDV(6) – U.S. — — — 1 Other HBV/HDV(6) – Europe 19 13 50 41 20 14 51 42 Total HBV/HDV – U.S. 116 131 299 311 Total HBV/HDV – Europe 34 28 96 85 Total HBV/HDV – Other International 119 106 363 337 269 264 758 733 Total Liver Disease – U.S. 376 413 1,051 1,056 Total Liver Disease – Europe 119 170 390 417 Total Liver Disease – Other International 211 204 652 631 706 788 2,093 2,104 Veklury Veklury – U.S. 258 336 607 1,179 Veklury – Europe 65 130 227 560 Veklury – Other International 313 458 630 1,166 636 925 1,465 2,905 Other AmBisome – U.S. 12 9 39 48 AmBisome – Europe 63 63 192 192 AmBisome – Other International 39 33 150 140 115 105 381 380 Letairis – U.S. 36 43 106 135 Other(7) – U.S. 34 28 91 91 Other(7) – Europe 9 11 31 52 Other(7) – Other International 23 13 49 35 65 52 171 178 Total Other – U.S. 82 80 236 275 Total Other – Europe 72 75 224 244 Total Other – Other International 62 46 199 174 216 200 658 693 Total product sales – U.S. 4,985 4,900 14,196 13,482 Total product sales – Europe 1,017 1,064 3,069 3,281 Total product sales – Other International 992 1,013 2,599 2,887 $ 6,994 $ 6,978 $ 19,864 $ 19,650 (1) Represents Gilead’s revenue from cobicistat (“C”), FTC and TAF in Symtuza (darunavir/C/FTC/TAF), a fixed dose combination product commercialized by Janssen Sciences Ireland Unlimited Company. (2) Includes Atripla, Emtriva, Sunlenca and Tybost. (3) Amounts consist of sales of Harvoni and the authorized generic version of Harvoni sold by Gilead’s separate subsidiary, Asegua Therapeutics LLC. (4) Amounts consist of sales of Epclusa and the authorized generic version of Epclusa sold by Gilead’s separate subsidiary, Asegua Therapeutics LLC. (5) Includes Vosevi and Sovaldi. (6) Includes Hepcludex and Hepsera. (7) Includes Cayston, Jyseleca, Ranexa and Zydelig.