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Achieved Fourth Quarter and Full Year 2023 Global Net Product Revenues of $346 Million and $1,241 Million, Respectively, Representing 39% Annual Growth Compared to 2022 Company Announces Updated Statistical Analysis Plan and Timing for HELIOS-B Phase 3 Study of Vutrisiran Announces U.S. FDA Clearance to Initiate Multiple-Dosing in ALN-APP Phase 1 Study Provides 2024 Combined Net Product Revenue Guidance of $1,400 Million to $1,500 Million, in Addition to Collaboration and Royalty Revenue and Operating Expense Guidance CAMBRIDGE, Mass.--(BUSINESS WIRE)-- Alnylam Pharmaceuticals, Inc. (Nasdaq: ALNY), the leading RNAi therapeutics company, today reported its consolidated financial results for the fourth quarter and full year ended December 31, 2023 and reviewed recent business highlights. “2023 was a year of strong execution at Alnylam. We delivered robust product revenue growth across our four wholly-owned commercial medicines, with $1.24 billion in global net product revenues, and achieved over 5,000 patients now being treated with an Alnylam commercial medicine. We also extended our leadership in RNAi with the first clinical demonstration of gene silencing in the human brain using an RNAi therapeutic, strengthened our business for the future through a landmark partnership with Roche and delivered solid financial performance,” said Yvonne Greenstreet, MBChB, Chief Executive Officer of Alnylam. “Looking ahead to 2024, we are excited for a number of important milestones across the pipeline, including results from the HELIOS-B Phase 3 study of vutrisiran as well as the KARDIA-2 Phase 2 study of zilebesiran, and six clinical study starts. This progress sets us up for delivering on our Alnylam P5x25 goals of becoming a top-tier biotech company delivering sustained innovation and exceptional financial results.” Fourth Quarter 2023 and Recent Significant Corporate Highlights Commercial Performance Total TTR: ONPATTRO® (patisiran) & AMVUTTRA® (vutrisiran) Achieved global net product revenues for ONPATTRO and AMVUTTRA for the fourth quarter of $79 million and $175 million, respectively, representing 10% total TTR quarterly growth compared to Q3 2023, and full year 2023 revenues of $355 million and $558 million, respectively, representing 40% total TTR annual growth compared to full year 2022. Attained over 4,060 hATTR amyloidosis patients with polyneuropathy worldwide on commercial treatment with ONPATTRO or AMVUTTRA as of December 31, 2023. Total Ultra-Rare: GIVLAARI® (givosiran) & OXLUMO® (lumasiran) Achieved global net product revenues for GIVLAARI and OXLUMO for the fourth quarter of $59 million and $33 million, respectively, representing 11% total Ultra-Rare quarterly growth compared to Q3 2023, and full year 2023 revenues of $219 million and $110 million, respectively, representing 35% total Ultra-Rare annual growth compared to full year 2022. Attained over 650 patients on commercial GIVLAARI and over 430 patients on commercial OXLUMO worldwide as of December 31, 2023. R&D Highlights Alnylam announces today updates to the statistical analysis plan for the HELIOS-B Phase 3 study of vutrisiran in patients with ATTR amyloidosis with cardiomyopathy. These will include updates to the primary and secondary endpoint structure, as well as study exposure. Topline results are expected to be available in late June or early July. Details will be discussed on the conference call this morning. Published results from Phase 3 APOLLO-B study of patisiran in the New England Journal of Medicine. Presented positive initial Phase 1 results with ALN-TTRsc04 demonstrating rapid knockdown achieved by a single dose with mean serum TTR reduction up to 97%, with durability supporting potential for annual dosing and an encouraging safety profile. Presented positive results from the KARDIA-1 Phase 2 dose-ranging study of zilebesiran, an investigational RNAi therapeutic in development to treat hypertension patients at high cardiovascular risk, during the American Heart Association (AHA) Scientific Sessions. Announces today that the U.S. Food and Drug Administration (FDA) has provided clearance to initiate the multiple-dose part (Part B) of the ongoing Phase 1 study of ALN-APP, an investigational RNAi therapeutic targeting amyloid precursor protein (APP) in development for the treatment of Alzheimer’s disease and cerebral amyloid angiopathy. The FDA has confirmed that multiple-dosing in the Phase 1 study may proceed at doses up to 180 mg given every six months, which covers all dose regimens planned to be explored in Part B. A partial clinical hold remains for higher or more frequent dosing regimens. Reported updated positive interim results for the ongoing single ascending dose portion of the Phase 1 study of ALN-APP in patients with early-onset Alzheimer’s disease at the 2023 Alzheimer’s Association International Conference and at the 16th Clinical Trials in Alzheimer’s Disease conference. Presented positive initial Phase 1 results with ALN-KHK demonstrating robust target engagement and an encouraging safety profile, supporting continued development as a novel treatment for type 2 diabetes mellitus. Filed an Investigational New Drug (IND) application for ALN-BCAT, an investigational RNAi therapeutic targeting β-catenin in development for the treatment of hepatocellular carcinoma. Sanofi presented positive results from the ATLAS-OLE Phase 3 extension study of fitusiran, demonstrating a substantially improved safety pro consistent bleed protection in people with hemophilia A or B, with or without inhibitors. Specifically, the risk of thrombosis was reduced, with rates comparable to those reported in the general hemophilia population. Sanofi expects to submit a New Drug Application (NDA) to the FDA in 2024. Additional Business Updates Ranked #1 on Boston Globe’s Top Places to Work list for 2023 in the “Largest Employer” category. Recognized by Science magazine as a Top Employer for the fifth consecutive year. Upcoming Events Alnylam announces today that results from the KARDIA-2 Phase 2 study of zilebesiran will be presented in a Late-Breaker presentation at the American College of Cardiology Scientific Sessions 2024 on April 7, 2024 in Atlanta, Georgia. In early 2024, Alnylam intends to: Report topline results from the HELIOS-B Phase 3 study of vutrisiran in late June or early July. Initiate the KARDIA-3 Phase 2 study of zilebesiran. Initiate a Phase 2 study of ALN-APP in patients with cerebral amyloid angiopathy. Initiate Part B of the Phase 1 study of ALN-KHK. Initiate a Phase 1 study of ALN-BCAT. Financial Highlights for the Fourth Quarter and Year End 2023 Three Months Ended December 31, Twelve Months Ended December 31, (In thousands, except per share amounts) 2023 2022 2023 2022 Net product revenues $ 346,288 $ 261,675 $ 1,241,474 $ 894,329 Net revenue from collaborations $ 76,407 $ 70,645 $ 546,185 $ 134,912 Royalty revenue $ 17,023 $ 2,715 $ 40,633 $ 8,177 GAAP Operating loss $ (116,404 ) $ (188,614 ) $ (282,175 ) $ (785,072 ) Non-GAAP Operating loss $ (74,410 ) $ (145,847 ) $ (60,495 ) $ (554,423 ) GAAP Net loss $ (137,870 ) $ (207,493 ) $ (440,242 ) $ (1,131,156 ) Non-GAAP Net loss $ (96,643 ) $ (171,522 ) $ (201,618 ) $ (790,609 ) GAAP Net loss per common share - basic and diluted $ (1.10 ) $ (1.68 ) $ (3.52 ) $ (9.30 ) Non-GAAP Net loss per common share - basic and diluted $ (0.77 ) $ (1.39 ) $ (1.61 ) $ (6.50 ) For an explanation of our use of non-GAAP financial measures refer to the "Use of Non-GAAP Financial Measures" section later in this press release and for a reconciliation of each non-GAAP financial measure to the most comparable GAAP measures, see the table at the end of this press release. Net Product Revenues Three Months Ended December 31, Twelve Months Ended December 31, (In thousands) 2023 2022 2023 2022 ONPATTRO net product revenues $ 79,006 $ 122,221 $ 354,546 $ 557,608 AMVUTTRA net product revenues 175,254 68,566 557,838 93,795 Total TTR net product revenues 254,260 190,787 912,384 651,403 GIVLAARI net product revenues 59,298 47,058 219,251 173,144 OXLUMO net product revenues 32,730 23,830 109,839 69,782 Total net product revenues $ 346,288 $ 261,675 $ 1,241,474 $ 894,329 Year over Year % Growth Three Months Ended December 31, 2023 Twelve Months Ended December 31, 2023 As Reported At CER* As Reported At CER* Total TTR net product revenues 33 % 31 % 40 % 40 % GIVLAARI net product revenues 26 % 24 % 27 % 26 % OXLUMO net product revenues 37 % 32 % 57 % 55 % Total net product revenues 32 % 30 % 39 % 39 % * CER = Constant Exchange Rate, representing growth calculated as if the exchange rates had remained unchanged from those used in 2022. CER is a non-GAAP measure. Net product revenues increased 32% and 39% at actual currency during the three and twelve months ended December 31, 2023, respectively, compared to the same periods in 2022, and 30% and 39% at CER, respectively. The increases are primarily due to increased patients on our commercial TTR therapies driven by the launch of AMVUTTRA in the third quarter of 2022 as well as increased patients on GIVLAARI and OXLUMO therapies. Net Revenues from Collaborations Net revenues from collaborations increased 8% and 305% during the three and twelve months ended December 31, 2023, respectively, as compared to the same periods in 2022, primarily due to revenue recognized under our Collaboration and License Agreement with Roche, as executed in July 2023, and revenue recognized under our Novartis Collaboration Agreement associated with the achievement of specified commercialization and regulatory milestones. Operating Expenses Three Months Ended December 31, Twelve Months Ended December 31, (In thousands) 2023 2022 2023 2022 Cost of goods sold $ 71,975 $ 46,172 $ 268,216 $ 140,174 Cost of goods sold as a percentage of net product revenues 20.8 % 17.6 % 21.6 % 15.7 % Cost of collaborations and royalties $ 13,883 $ 5,094 $ 42,190 $ 28,643 GAAP research and development expenses $ 272,141 $ 262,039 $ 1,004,415 $ 883,015 Non-GAAP research and development expenses $ 253,056 $ 245,095 $ 907,142 $ 790,854 GAAP selling, general and administrative expenses $ 198,123 $ 210,344 $ 795,646 $ 770,658 Non-GAAP selling, general and administrative expenses $ 175,214 $ 184,521 $ 671,239 $ 632,170 Cost of Goods Sold Cost of goods sold as a percentage of net product revenues increased during the three and twelve months ended December 31, 2023, respectively, as compared to the same periods in 2022, primarily due to increased volume and rate of royalties payable on net sales of AMVUTTRA associated with tiered royalty percentages, in addition to increased excess and obsolete charges primarily due to canceling manufacturing commitments and the impairment of ONPATTRO inventory that had been manufactured for future demand associated with the ATTR amyloidosis with cardiomyopathy indication for patisiran for which we did not receive regulatory approval. Research & Development (R&D) Expenses GAAP and non-GAAP R&D expenses increased during the three and twelve months ended December 31, 2023, compared to the same periods in 2022, primarily due to increased headcount and infrastructure expenses to support our R&D pipeline, development expenses associated with the KARDIA-1 and KARDIA-2 zilebesiran Phase 2 studies, and manufacturing and research related expenses associated with our pre-clinical and developmental activities. GAAP R&D expenses further increased during the twelve month period due to increased stock-based compensation expense related to the accounting for certain performance-based awards during the period. Selling, General & Administrative (SG&A) Expenses GAAP and non-GAAP SG&A expenses decreased during the three months ended December 31, 2023, compared to the same period in 2022, primarily due to increased legal expenses in 2022 associated with the Patent Infringement Lawsuits we filed in March 2022 and the Department of Justice investigation, which closed in August 2023, and increased expenses in support of the global launch of AMVUTTRA in the third quarter of 2022. GAAP and non-GAAP SG&A expenses increased during the twelve months ended December 31, 2023, compared to the same period in 2022, primarily due to increased headcount and other strategic investments in support of the global launch of AMVUTTRA and other expenses to support our strategic growth. Other Financial Highlights Cash, cash equivalents and marketable securities were $2.44 billion as of December 31, 2023, compared to $2.19 billion as of December 31, 2022, with the increase primarily due to the receipts of a $310 million upfront payment from Roche in connection with our partnership to co-develop and co-commercialize zilebesiran, a $100 million payment from Regeneron in connection with the achievement of certain criteria during early clinical development for our CNS program, ALN-APP, and nearly $150 million from employee option award exercises, offset by our operating loss for the year. A reconciliation of our GAAP to non-GAAP results for the current quarter is included in the tables at the end of this press release. 2024 Financial Guidance Full year December 31, 2024 financial guidance consists of the following: Combined net product revenues for AMVUTTRA, ONPATTRO, GIVLAARI and OXLUMO1 $1,400 million – $1,500 million Net Product Revenue Growth vs. 2023 at reported Fx rates1 13% – 21% Net Product Revenue Growth vs. 2023 at constant exchange rates* 13% – 21% Net revenues from collaborations and royalties $325 million – $425 million GAAP R&D and SG&A expenses $1,900 million - $2,050 million Non-GAAP R&D and SG&A expenses2 $1,675 million – $1,775 million 1 Uses January 31, 2024 FX rates including: 1 EUR = 1.08 USD and 1 USD = 147 JPY 2 Primarily excludes $225-$275 million of stock-based compensation expense from estimated GAAP R&D and SG&A expenses. * CER = Constant Exchange Rate, representing growth calculated as if the exchange rates had remained unchanged from those used in the twelve months ended December 31, 2023. CER is a non-GAAP measure. Use of Non-GAAP Financial Measures This press release contains non-GAAP financial measures, including expenses adjusted to exclude certain non-cash expenses and non-recurring gains outside the ordinary course of the Company’s business. These measures are not in accordance with, or an alternative to, GAAP, and may be different from non-GAAP financial measures used by other companies. The items included in GAAP presentations but excluded for purposes of determining non-GAAP financial measures for the periods presented in this press release are, as applicable, stock-based compensation expenses, realized and unrealized (gains) losses on marketable equity securities and loss on the extinguishment of debt. The Company has excluded the impact of stock-based compensation expense, which may fluctuate from period to period based on factors including the variability associated with performance-based grants for stock options and restricted stock units and changes in the Company’s stock price, which impacts the fair value of these awards. The Company has excluded the impact of the realized and unrealized (gains) losses on marketable equity securities because the Company does not believe these adjustments accurately reflect the performance of the Company’s ongoing operations for the period in which such gains or losses are reported, as their sole purpose is to adjust amounts on the balance sheet. The Company has excluded the loss on the extinguishment of debt because the Company believes the item is a non-recurring transaction outside the ordinary course of the Company’s business. Percentage changes in revenue growth at CER are presented excluding the impact of changes in foreign currency exchange rates for investors to understand the underlying business performance. The current period’s foreign currency revenue values are converted into U.S. dollars using the average exchange rates from the prior period. The Company believes the presentation of non-GAAP financial measures provides useful information to management and investors regarding the Company’s financial condition and results of operations. When GAAP financial measures are viewed in conjunction with non-GAAP financial measures, investors are provided with a more meaningful understanding of the Company’s ongoing operating performance and are better able to compare the Company’s performance between periods. In addition, these non-GAAP financial measures are among those indicators the Company uses as a basis for evaluating performance, allocating resources and planning and forecasting future periods. Non-GAAP financial measures are not intended to be considered in isolation or as a substitute for GAAP financial measures. A reconciliation between GAAP and non-GAAP measures is provided later in this press release. Conference Call Information Management will provide an update on the Company and discuss fourth quarter and full year 2023 results as well as expectations for the future via conference call on Thursday, February 15, 2024 at 8:30 am ET. To access the call, please register online at . Participants are requested to register at least 15 minutes before the start of the call. A replay of the call will be available two hours after the call and archived on the same web page for six months. A live audio webcast of the call will be available on the Investors section of the Company’s website at . An archived webcast will be available on the Alnylam website approximately two hours after the event. About ONPATTRO® (patisiran) ONPATTRO is an RNAi therapeutic that is approved in the United States and Canada for the treatment of adults with hATTR amyloidosis with polyneuropathy. ONPATTRO is also approved in the European Union, Switzerland and Brazil for the treatment of hATTR amyloidosis in adults with Stage 1 or Stage 2 polyneuropathy, and in Japan for the treatment of hATTR amyloidosis with polyneuropathy. ONPATTRO is an intravenously administered RNAi therapeutic targeting transthyretin (TTR). It is designed to target and silence TTR messenger RNA, thereby reducing the production of TTR protein before it is made. Reducing the pathogenic protein leads to a reduction in amyloid deposits in tissues. For more information about ONPATTRO, including full Prescribing Information, visit ONPATTRO.com. About AMVUTTRA® (vutrisiran) AMVUTTRA® (vutrisiran) is an RNAi therapeutic approved in the United States for the treatment of adults with hATTR amyloidosis with polyneuropathy. It is a double‑stranded small interfering RNA (siRNA) that targets mutant and wild‑type transthyretin (TTR) messenger RNA (mRNA). Using Alnylam’s Enhanced Stabilization Chemistry (ESC)-GalNAc-conjugate delivery platform, AMVUTTRA is designed for increased potency and high metabolic stability to allow for subcutaneous injection once every three months (quarterly). Results from the pivotal HELIOS-A Phase 3 study demonstrate AMVUTTRA rapidly reduces serum TTR levels, has the potential to reverse neuropathy impairment relative to baseline and improves other key measures of disease burden relative to external placebo in patients with the polyneuropathy of hATTR amyloidosis. For more information about AMVUTTRA, including the full U.S. Prescribing Information, visit AMVUTTRA.com. About GIVLAARI® (givosiran) GIVLAARI (givosiran) is an RNAi therapeutic targeting aminolevulinic acid synthase 1 (ALAS1) approved in the United States and Brazil for the treatment of adults with acute hepatic porphyria (AHP). GIVLAARI is also approved in the European Union for the treatment of AHP in adults and adolescents aged 12 years and older. In the pivotal study, givosiran was shown to significantly reduce the rate of porphyria attacks that required hospitalizations, urgent healthcare visits or intravenous hemin administration at home compared to placebo. GIVLAARI is Alnylam’s first commercially available therapeutic based on its Enhanced Stabilization Chemistry ESC-GalNAc conjugate technology to increase potency and durability. GIVLAARI is administered via subcutaneous injection once monthly at a dose based on actual body weight and should be administered by a healthcare professional. GIVLAARI works by specifically reducing elevated levels of ALAS1 messenger RNA (mRNA), leading to reduction of toxins associated with attacks and other disease manifestations of AHP. For more information about GIVLAARI, including the full U.S. Prescribing Information, visit GIVLAARI.com. About OXLUMO® (lumasiran) OXLUMO (lumasiran) is an RNAi therapeutic targeting hydroxyacid oxidase 1 (HAO1). HAO1 encodes glycolate oxidase (GO). Thus, by silencing HAO1 and depleting the GO enzyme, OXLUMO inhibits production of oxalate – the metabolite that directly contributes to the pathophysiology of PH1. OXLUMO utilizes Alnylam’s Enhanced Stabilization Chemistry (ESC)-GalNAc-conjugate technology, which enables subcutaneous dosing with increased potency and durability and a wide therapeutic index. OXLUMO has received regulatory approvals from the U.S. Food and Drug Administration (FDA) for the treatment of primary hyperoxaluria type 1 (PH1) to lower urinary and plasma oxalate levels in pediatric and adult patients and from the European Medicines Agency (EMA) for the treatment of PH1 in all age groups. In the pivotal ILLUMINATE-A study, OXLUMO was shown to significantly reduce levels of urinary oxalate relative to placebo, with the majority of patients reaching normal or near-normal levels. In the ILLUMINATE-B pediatric Phase 3 study, OXLUMO demonstrated an efficacy and safety pro to that observed in ILLUMINATE-A. In the ILLUMINATE-C study, OXLUMO resulted in substantial reductions in plasma oxalate in patients with advanced PH1. Across all three studies, injection site reactions (ISRs) were the most common drug-related adverse reaction. OXLUMO is administered via subcutaneous injection once monthly for three months, then once quarterly beginning one month after the last loading dose at a dose based on actual body weight. For patients who weigh less than 10 kg, ongoing dosing remains monthly. OXLUMO should be administered by a healthcare professional. For more information about OXLUMO, including the full U.S. Prescribing Information, visit OXLUMO.com. About LNP Technology Alnylam has licenses to Arbutus Biopharma LNP intellectual property for use in RNAi therapeutic products using LNP technology. About RNAi RNAi (RNA interference) is a natural cellular process of gene silencing that represents one of the most promising and rapidly advancing frontiers in biology and drug development today. Its discovery has been heralded as “a major scientific breakthrough that happens once every decade or so,” and was recognized with the award of the 2006 Nobel Prize for Physiology or Medicine. By harnessing the natural biological process of RNAi occurring in our cells, a new class of medicines known as RNAi therapeutics is now a reality. Small interfering RNA (siRNA), the molecules that mediate RNAi and comprise Alnylam's RNAi therapeutic platform, function upstream of today’s medicines by potently silencing messenger RNA (mRNA) – the genetic precursors – that encode for disease-causing or disease pathway proteins, thus preventing them from being made. This is a revolutionary approach with the potential to transform the care of patients with genetic and other diseases. About Alnylam Pharmaceuticals Alnylam Pharmaceuticals (Nasdaq: ALNY) has led the translation of RNA interference (RNAi) into a whole new class of innovative medicines with the potential to transform the lives of people afflicted with rare and prevalent diseases with unmet need. Based on Nobel Prize-winning science, RNAi therapeutics represent a powerful, clinically validated approach yielding transformative medicines. Since its founding in 2002, Alnylam has led the RNAi Revolution and continues to deliver on a bold vision to turn scientific possibility into reality. Alnylam’s commercial RNAi therapeutic products are ONPATTRO® (patisiran), AMVUTTRA® (vutrisiran), GIVLAARI® (givosiran), OXLUMO® (lumasiran), and Leqvio® (inclisiran), which is being developed and commercialized by Alnylam’s partner, Novartis. Alnylam has a deep pipeline of investigational medicines, including multiple product candidates that are in late-stage development. Alnylam is executing on its “Alnylam P5x25” strategy to deliver transformative medicines in both rare and common diseases benefiting patients around the world through sustainable innovation and exceptional financial performance, resulting in a leading biotech profile. Alnylam is headquartered in Cambridge, MA. For more information about our people, science and pipeline, please visit and engage with us on X (formerly Twitter) at @Alnylam, or on LinkedIn, Facebook, or Instagram. Alnylam Forward Looking Statements This press release contains forward-looking statements within the meaning of Section 27A of the Securities Act of 1933 and Section 21E of the Securities Exchange Act of 1934. All statements other than historical statements of fact regarding Alnylam’s expectations, beliefs, goals, plans or prospects including, without limitation, statements regarding Alnylam’s aspiration to become a top-tier biotech company, the potential for Alnylam to identify new potential drug development candidates and advance its research and development programs, Alnylam’s ability to obtain approval for new commercial products or additional indications for its existing commercial products, and Alnylam’s projected commercial and financial performance, including the expected range of net product revenues and net revenues from collaborations and royalties for 2024, the expected range of aggregate annual GAAP and non-GAAP R&D and SG&A expenses for 2024, the expected timing of topline data from the HELIOS-B Phase 3 clinical study, and the planned achievement of its “Alnylam P5x25” strategy, should be considered forward-looking statements. Actual results and future plans may differ materially from those indicated by these forward-looking statements as a result of various important risks, uncertainties and other factors, including, without limitation, risks and uncertainties relating to: Alnylam’s ability to successfully execute on its “Alnylam P5x25” strategy; Alnylam’s ability to discover and develop novel drug candidates and delivery approaches and successfully demonstrate the efficacy and safety of its product candidates; the pre-clinical and clinical results for Alnylam’s product candidates, including vutrisiran, zilebesiran, and ALN-APP; actions or advice of regulatory agencies and Alnylam’s ability to obtain and maintain regulatory approval for its product candidates, including vutrisiran, as well as favorable pricing and reimbursement; successfully launching, marketing and selling Alnylam’s approved products globally; delays, interruptions or failures in the manufacture and supply of Alnylam’s product candidates or its marketed products; obtaining, maintaining and protecting intellectual property; Alnylam’s ability to successfully expand the approved indications for AMVUTTRA in the future; Alnylam’s ability to manage its growth and operating expenses through disciplined investment in operations and its ability to achieve a self-sustainable financial pro the future without the need for future equity financing; the direct or indirect impact of the COVID-19 global pandemic or any future pandemic on Alnylam’s business, results of operations and financial condition; Alnylam’s ability to maintain strategic business collaborations; Alnylam’s dependence on third parties for the development and commercialization of certain products, including Roche, Novartis, Sanofi, Regeneron and Vir; the outcome of litigation; the risk of future government investigations; and unexpected expenditures; as well as those risks and uncertainties more fully discussed in the “Risk Factors” filed with Alnylam’s most recent Quarterly Report on Form 10-Q filed with the Securities and Exchange Commission (SEC) and in other filings that Alnylam makes with the SEC. In addition, any forward-looking statements represent Alnylam's views only as of today and should not be relied upon as representing its views as of any subsequent date. Alnylam explicitly disclaims any obligation, except to the extent required by law, to update any forward-looking statements. This release discusses investigational RNAi therapeutics and uses of previously approved RNAi therapeutics in development and is not intended to convey conclusions about efficacy or safety as to those investigational therapeutics or uses. There is no guarantee that any investigational therapeutics or expanded uses of commercial products will successfully complete clinical development or gain health authority approval. ALNYLAM PHARMACEUTICALS, INC. CONSOLIDATED BALANCE SHEETS (In thousands, except per share amounts) December 31, 2023 December 31, 2022 ASSETS Current assets: Cash and cash equivalents $ 812,688 $ 866,394 Marketable debt securities 1,615,516 1,297,890 Marketable equity securities 11,178 28,122 Accounts receivable, net 327,787 237,963 Inventory 89,146 128,962 Prepaid expenses and other current assets 126,382 132,916 Total current assets 2,982,697 2,692,247 Property, plant and equipment, net 526,057 523,494 Operating lease right-of-use assets 199,732 215,136 Restricted investments 49,391 49,390 Other assets 72,003 66,092 Total assets $ 3,829,880 $ 3,546,359 LIABILITIES AND STOCKHOLDERS’ DEFICIT Current liabilities: Accounts payable $ 55,519 $ 98,094 Accrued expenses 713,013 545,460 Operating lease liability 41,510 41,967 Deferred revenue 102,753 42,105 Liability related to the sale of future royalties 54,991 40,289 Total current liabilities 967,786 767,915 Operating lease liability, net of current portion 243,101 261,339 Deferred revenue, net of current portion 188,175 193,791 Convertible debt 1,020,776 1,016,942 Liability related to the sale of future royalties, net of current portion 1,322,248 1,252,015 Other liabilities 308,438 212,580 Total liabilities 4,050,524 3,704,582 Commitments and contingencies (Note 13) Stockholders’ deficit: Preferred stock, $0.01 par value per share, 5,000 shares authorized and no shares issued and outstanding as of December 31, 2023 and December 31, 2022 — — Common stock, $0.01 par value per share, 250,000 shares authorized as of December 31, 2023 and December 31, 2022, respectively; 125,794 shares issued and outstanding as of December 31, 2023; 123,925 shares issued and outstanding as of December 31, 2022 1,259 1,240 Additional paid-in capital 6,811,063 6,454,540 Accumulated other comprehensive loss (23,375 ) (44,654 ) Accumulated deficit (7,009,591 ) (6,569,349 ) Total stockholders’ deficit (220,644 ) (158,223 ) Total liabilities and stockholders’ deficit $ 3,829,880 $ 3,546,359 This selected financial information should be read in conjunction with the consolidated financial statements and notes thereto included in Alnylam’s Annual Report on Form 10-K which includes the audited financial statements for the year ended December 31, 2023. ALNYLAM PHARMACEUTICALS, INC. CONSOLIDATED STATEMENTS OF OPERATIONS (In thousands, except per share amounts) Three Months Ended December 31, Twelve Months Ended December 31, 2023 2022 2023 2022 (Unaudited) (Unaudited) Revenues: Net product revenues $ 346,288 $ 261,675 $ 1,241,474 $ 894,329 Net revenues from collaborations 76,407 70,645 546,185 134,912 Royalty revenue 17,023 2,715 40,633 8,177 Total revenues 439,718 335,035 1,828,292 1,037,418 Operating costs and expenses: Cost of goods sold 71,975 46,172 268,216 140,174 Cost of collaborations and royalties 13,883 5,094 42,190 28,643 Research and development 272,141 262,039 1,004,415 883,015 Selling, general and administrative 198,123 210,344 795,646 770,658 Total operating costs and expenses 556,122 523,649 2,110,467 1,822,490 Loss from operations (116,404 ) (188,614 ) (282,175 ) (785,072 ) Other (expense) income: Interest expense (31,338 ) (29,913 ) (121,221 ) (155,968 ) Interest income 30,406 14,077 95,561 24,808 Other expense, net (20,351 ) (2,571 ) (125,682 ) (134,175 ) Loss on the extinguishment of debt — — — (76,586 ) Total other expense, net (21,283 ) (18,407 ) (151,342 ) (341,921 ) Loss before income taxes (137,687 ) (207,021 ) (433,517 ) (1,126,993 ) Provision for income taxes (183 ) (472 ) (6,725 ) (4,163 ) Net loss $ (137,870 ) $ (207,493 ) $ (440,242 ) $ (1,131,156 ) Net loss per common share — basic and diluted $ (1.10 ) $ (1.68 ) $ (3.52 ) $ (9.30 ) Weighted-average common shares used to compute basic and diluted net loss per common share 125,613 123,266 124,906 121,689 ALNYLAM PHARMACEUTICALS, INC. RECONCILIATION OF SELECTED GAAP MEASURES TO NON-GAAP MEASURES (In thousands, except per share amounts) (Unaudited) Three Months Ended December 31, Twelve Months Ended December 31, 2023 2022 2023 2022 Reconciliation of GAAP to Non-GAAP research and development: GAAP Research and development $ 272,141 $ 262,039 $ 1,004,415 $ 883,015 Less: Stock-based compensation expenses (19,085 ) (16,944 ) (97,273 ) (92,161 ) Non-GAAP Research and development $ 253,056 $ 245,095 $ 907,142 $ 790,854 Reconciliation of GAAP to Non-GAAP selling, general and administrative: GAAP Selling, general and administrative $ 198,123 $ 210,344 $ 795,646 $ 770,658 Less: Stock-based compensation expenses (22,909 ) (25,823 ) (124,407 ) (138,488 ) Non-GAAP Selling, general and administrative $ 175,214 $ 184,521 $ 671,239 $ 632,170 Reconciliation of GAAP to Non-GAAP operating loss: GAAP operating loss $ (116,404 ) $ (188,614 ) $ (282,175 ) $ (785,072 ) Add: Stock-based compensation expenses 41,994 42,767 221,680 230,649 Non-GAAP Operating loss $ (74,410 ) $ (145,847 ) $ (60,495 ) $ (554,423 ) Reconciliation of GAAP to Non-GAAP Other (expense) income: GAAP Total other expense, net $ (21,283 ) $ (18,407 ) $ (151,342 ) $ (341,921 ) (Less) Add: Realized and unrealized (gain) loss on marketable equity securities (767 ) (6,796 ) 16,944 33,312 Add: Loss on the extinguishment of debt — — — 76,586 Non-GAAP Other expense, net $ (22,050 ) $ (25,203 ) $ (134,398 ) $ (232,023 ) Reconciliation of GAAP to Non-GAAP net loss: GAAP Net loss $ (137,870 ) $ (207,493 ) $ (440,242 ) $ (1,131,156 ) Add: Stock-based compensation expenses 41,994 42,767 221,680 230,649 (Less) Add: Realized and unrealized (gain) loss on marketable equity securities (767 ) (6,796 ) 16,944 33,312 Add: Loss on the extinguishment of debt — — — 76,586 Non-GAAP Net loss $ (96,643 ) $ (171,522 ) $ (201,618 ) $ (790,609 ) Reconciliation of GAAP to Non-GAAP net loss per common share-basic and diluted: GAAP Net loss per common share - basic and diluted $ (1.10 ) $ (1.68 ) $ (3.52 ) $ (9.30 ) Add: Stock-based compensation expenses 0.33 0.35 1.77 1.90 (Less) Add: Realized and unrealized (gain) loss on marketable equity securities (0.01 ) (0.06 ) 0.14 0.27 Add: Loss on the extinguishment of debt — — — 0.63 Non-GAAP Net loss per common share - basic and diluted $ (0.77 ) $ (1.39 ) $ (1.61 ) $ (6.50 ) Please note that the figures presented above may not sum exactly due to rounding ALNYLAM PHARMACEUTICALS, INC. RECONCILIATION OF GAAP TO NON-GAAP PRODUCT REVENUE GROWTH AT CONSTANT CURRENCY (Unaudited) December 31, 2023 Three Months Ended Twelve Months Ended Total TTR net product revenue growth, as reported 33 % 40 % Add: Impact of foreign currency translation (2 ) — Total TTR net product revenue growth at constant currency 31 % 40 % GIVLAARI net product revenue growth, as reported 26 % 27 % Add: Impact of foreign currency translation (2 ) (1 ) GIVLAARI net product revenue growth at constant currency 24 % 26 % OXLUMO net product revenue growth, as reported 37 % 57 % Add: Impact of foreign currency translation (5 ) (2 ) OXLUMO net product revenue growth at constant currency 32 % 55 % Total net product revenue growth, as reported 32 % 39 % Add: Impact of foreign currency translation (2 ) — Total net product revenue growth at constant currency 30 % 39 % Total revenue growth, as reported 31 % 76 % Add: Impact of foreign currency translation (2 ) — Total revenue growth at constant currency 29 % 76 % View source version on businesswire.com: Contacts Alnylam Pharmaceuticals, Inc. Christine Regan Lindenboom (Investors and Media) 617-682-4340 Josh Brodsky (Investors) 617-551-8276 Source: Alnylam Pharmaceuticals, Inc. View this news release online at:

嘉宾阵容以及授课话题已曝光，点击图片查询，合作热线：王晨 180 1628 8769截止目前，（不统计DNA疫苗和mRNA疫苗在内）目前全球已有54款基因治疗药物获批上市，其中体外基因疗法17款；基于病毒载体的体内基因疗法16款；小核酸药物19款；其它2款。随着越来越多的基因治疗产品的上市，以及基因治疗技术的快速发展，基因治疗即将迎来高速发展期。FDA 曾在多个场合表示，他们预计到2025年，每年将批准10至20种细胞基因疗法。基因是控制性状的基本遗传单位，除某些病毒的基因由RNA构成以外，多数生物的基因由DNA构成。有机体的大多数疾病是由基因与环境相互作用而引发的，通过基因疗法可以从本质上治愈或缓解多种疾病，基因治疗被认为是医学和药学领域的一次革命，广义的基因治疗药物包括基于DNA修饰的DNA药物（例如基于病毒载体的体内基因治疗药物、体外基因治疗药物、裸质粒药物等）与RNA药物（例如反义寡核苷酸药物、siRNA药物和mRNA基因治疗等）；狭义的基因治疗药物主要包括包括质粒DNA药物、基于病毒载体的基因治疗药物、基于细菌载体的基因治疗药物、基因编辑系统和体外基因修饰的细胞治疗药物等。基因治疗的分类（图片来源：生物经纬）本文列举了（不包括DNA疫苗和mRNA疫苗在内）已获批上市的54款基因治疗药物（若不包括小核酸药物等在内，狭义上共35款）。1、体外基因疗法（1）Strimvelis公司：由葛兰素史克（GSK）公司研发。上市时间：2016年5月被欧盟批准上市。适应症：用于重症联合免疫缺陷症（简称SCID）治疗。备注：该疗法大致流程是首先获得患者自身的造血干细胞，在体外进行扩增培养，之后利用逆转录病毒将功能性ADA（腺苷脱氨酶）基因拷贝导入其造血干细胞，最后将修饰后的造血干细胞回输到本人体内。临床结果显示，Strimvelis治疗的ADA-SCID患者3年存活率为100%。（2）Zalmoxis公司：由意大利MolMed公司生产。上市时间：2016年获欧盟有条件上市许可。适应症：用于造血干细胞移植后患者免疫系统的辅助治疗。备注：Zalmoxis是一款经逆转录病毒载体改造的同种异体T细胞自杀基因免疫疗法，该方法使用逆转录病毒载体对同种异体来源的T细胞进行基因改造，使基因修饰后的T细胞表达1NGFR和HSV-TK Mut2自杀基因，使得人们可以随时使用更昔洛韦（ganciclovir）药物杀死引起不良免疫反应的T细胞，防止可能出现的GVHD进一步恶化，为半相合HSCT患者术后免疫功能重建保驾护航。（3）Invossa-K公司：由TissueGene公司研发。上市时间：2017年7月在韩国获得批准上市。适应症：用于治疗退行性膝关节炎。备注：Invossa-K是一种涉及人软骨细胞的同种异体细胞基因疗法，同种异体细胞经体外基因修饰，修饰后的细胞经关节内注射给药后可表达分泌转化生长因子β1（TGF-β1），进而改善骨关节炎症状。临床结果显示，Invossa-K能够显著改善膝关节炎。于2019年被韩国药监部门取消许可证，因为该制造商错误地标记了所使用的成分。（4）Zynteglo公司：由美国蓝鸟生物（bluebird bio）公司研发。上市时间：2019年获欧盟批准上市，2022年8月获FDA批准在美国上市。适应症：用于治疗输血依赖性β-地中海贫血。备注：Zynteglo是一款慢病毒体外基因疗法，该疗法通过慢病毒载体，把正常的β-珠蛋白基因（βA-T87Q-珠蛋白基因）的功能性拷贝导入到从患者体内取出的造血干细胞中，再将这些基因修饰的自体造血干细胞回输到患者体内，一旦患者拥有正常的βA-T87Q-珠蛋白基因，他们就有可能产生正常的HbAT87Q蛋白，即可有效降低或消除输血需求。这是一种一次性疗法，旨在取代终身输血和终生药物，适用于12岁及以上的患者。（5）Skysona公司：由美国蓝鸟生物（bluebird bio）公司研发。上市时间：2021年7月获欧盟批准上市，2022年9月获FDA批准在美国上市。适应症：用于治疗早期脑肾上腺脑白质营养不良（CALD）。备注：Skysona基因疗法是唯一一个获批用于治疗早期脑肾上腺脑白质营养不良（CALD）的一次性基因疗法。Skysona（elivaldogene autotemcel，Lenti-D）是一种造血干细胞慢病毒体外基因疗法Lenti-D。疗法大致流程为：从患者体内取出自体造血干细胞，体外经搭载人ABCD1基因的慢病毒转导修饰后，回输给患者。用于治疗年龄在18岁以下、携带ABCD1基因突变、CALD患者。（6）Kymriah公司：由诺华（Novartis）研发。上市时间：2017年8月获FDA批准上市。适应症：治疗前体B细胞急性淋巴细胞白血病（ALL）和复发、难治性DLBCL。备注：Kymriah是一款慢病毒体外基因治疗药物，是全球首款获批上市的CAR-T疗法，靶向CD19，采用了4-1BB的共刺激因子。在美国定价为47.5万美元，在日本定价为31.3万美元。（7）Yescarta公司：由吉利德（GILD）公司旗下Kite Pharma开发。上市时间：2017年10月获FDA批准上市；复星凯特从Kite Pharma引进Yescarta技术，且获得授权后在中国进行生产，其产品奕凯达（阿基仑塞注射液），于2021年6月在国内获批上市。适应症：用于治疗复发或难治性大B细胞淋巴瘤。备注：Yescarta是一款逆转录病毒体外基因疗法，是全球第二款获批上市的CAR-T疗法，靶向CD19，采用了CD28的共刺激因子。在美国定价为37.3万美元。（8）Tecartus公司：由吉利德（GILD）公司开发。上市时间：2020年7月份获FDA批准上市。适应症：用于复发或难治性套细胞淋巴瘤。备注：Tecartus是一款靶向CD19的自体CAR-T细胞疗法，是全球第三款获批上市的CAR-T疗法。（9）Breyanzi公司：由百时美施贵宝（BMS）开发。上市时间：2021年2月获FDA批准上市。适应症：复发或难治性（R/R）大B细胞淋巴瘤（LBCL）。备注：Breyanzi是一款基于慢病毒的体外基因疗法，是全球第四款获批上市的CAR-T疗法，靶向CD19。Breyanzi的获批是百时美施贵宝在细胞免疫治疗领域的一个里程碑，该药物是百时美施贵宝在2019年以740亿美元收购Celgene时获得的。（10）Abecma公司：由百时美施贵宝（BMS）和蓝鸟生物（bluebird bio）共同开发。上市时间：2021年3月由FDA批准上市。适应症：复发或难治性多发性骨髓瘤。备注：Abecma是一款基于慢病毒的体外基因疗法，是全球首款靶向BCMA的CAR-T细胞疗法，是FDA批准的第五款CAR-T疗法。其药物原理是体外通过慢病毒介导的基因修饰在患者自体T细胞上表达嵌合BCMA的受体，输注该细胞基因药物前，患者先接受环磷酰胺和氟达拉滨两种化合物预处理，以清除患者体内未基因修饰的T细胞，随后回输修饰后的T细胞，修饰后的T细胞在患者体内寻找并杀死表达BCMA的癌细胞。（11）Libmeldy公司：由Orchard Therapeutics公司开发。上市时间：2020年12月份获欧盟批准上市。适应症：用于治疗异染性脑白质营养不良（metachromatic leukodystrophy，MLD）。备注：Libmeldy是一款基于慢病毒体外基因修饰的自体CD34+细胞的基因疗法。临床数据显示，单次静脉输注Libmeldy可以有效地改变早发型MLD的病程，未接受治疗的同年龄段患者则出现严重的运动和认知障碍。（12）倍诺达公司：由药明巨诺公司开发。上市时间：2021年9月获NMPA正式批复。适应症：治疗经过二线或以上系统性治疗后成人患者的复发或难治性大B细胞淋巴瘤（r/r LBCL）。备注：倍诺达是一款抗CD19的CAR-T基因疗法，也是药明巨诺公司的核心产品，是中国第二款获批的CAR-T产品，除了复发/难治性大B细胞淋巴瘤，药明巨诺公司还计划开发瑞基奥仑赛注射液用于治疗多种其它适应症，包括滤泡淋巴瘤（FL）、套细胞淋巴瘤（MCL）、慢性淋巴细胞白血病（CLL）、二线弥漫性大B细胞淋巴瘤（DLBCL）及急性淋巴细胞白血病（ALL）等。（13）CARVYKTI公司：传奇生物首款获批上市的产品。上市时间：2022年2月获FDA批准上市。适应症：治疗复发或难治性多发性骨髓瘤（R/R MM）。备注：CARVYKTI（西达基奥仑赛，ciltacabtagene autoleucel，简称Cilta-cel）是一款具有两种靶向B细胞成熟抗原（BCMA）单域抗体的CAR-T细胞免疫基因疗法，数据显示，CARVYKTI在既往接受过四种或者以上治疗（包括蛋白酶体抑制剂、免疫调节剂和抗CD38单克隆抗体）的复发或难治性多发性骨髓瘤患者中显示出高达98%的总缓解率。（14）福可苏（伊基奥仑赛注射液）公司：由驯鹿生物与信达生物共同开发。上市时间：2023年6月30日获NMPA批准上市，为国内第三款上市的CAR-T疗法。适应症：治疗复发或难治性多发性骨髓瘤成人患者。备注：福可苏是一款靶向BCMA的体外基因治疗药物，通过工程化的慢病毒基因载体修饰患者自体T细胞。据悉，该药物上市获批主要是基于一项1/2期注册性临床的研究结果，公布的相关数据显示，经过至少三线治疗的多发性骨髓瘤患者接受该疗法治疗后，患者的总体缓解率为94.9%，完全缓解率/严格意义的完全缓解率为58.2%，表现出来良好的有效性。（15）纳基奥仑赛注射液公司：由合源生物公司开发。上市时间：2023年11月8日获NMPA批准上市。适应症：用于治疗成人复发或难治性B细胞型急性淋巴细胞白血病（r/r B-ALL）。备注：纳基奥仑赛注射液是一款基于慢病毒载体的自体CAR-T产品，其具有全球独特的CD19 scFv（HI19a）结构，其作为中国第一款、全球第二款用于治疗成人急性淋巴细胞白血病的CAR-T产品填补了国内细胞疗法在这一疾病治疗领域的空白。在关键性临床研究中展示出持久的高缓解率和良好的安全性。（16）CASGEVY公司：由CRISPR Therapeutics和Vertex联合开发。上市时间：2023年12月8日获FDA批准上市，这是FDA批准上市的首款CRISPR基因编辑疗法。适应症：用于治疗12岁及以上患有复发性血管闭塞危象 (VOC) 的镰状细胞病 (SCD) 患者。备注：Exagamglogene Autotemcel（Exa-cel，CASGEVY™）是一种一次性单剂量细胞基因治疗药物，由经过CRISPR/Cas9介导的基因编辑的自体CD34+人类造血干细胞和祖细胞 (hHSPC) 组成，发挥基因编辑作用的CRISPR-Cas9蛋白与sgRNA在体外经电穿孔的方式被递送到患者的离体细胞内，CRISPR复合体会结合在位于BCL11A基因内含子上的红细胞特异性增强子中--转录因子GATA1的结合区域，在引入DSB后，利用NHEJ产生突变，破坏该增强子，降低BCL11A在红细胞中的转录水平，从而特异性提高HbF在红细胞中的表达，HbF水平的升高能够缓解或消除SCD患者的疼痛及血管闭塞性危象 (VOC)，并且能缓解输血依赖性β-地中海贫血（TDT）患者的输血需求，使TDT患者摆脱长期输血的困扰。（17）LYFGENIA公司：美国蓝鸟生物（bluebird bio）公司研发。上市时间：2023年12月8日获FDA批准上市。适应症：用于治疗12 岁及以上患有复发性血管闭塞危象 (VOC) 的镰状细胞病 (SCD) 患者。备注：LYFGENIA（lovotibeglogene autotemcel）是一款基于慢病毒载体的细胞基因疗法，通过慢病毒载体将功能性β珠蛋白基因永久添加到患者自身的造血干细胞（HSC）中，可持久产生具有抗镰状细胞特性的血红蛋白（HbAT87Q型），HbAT87Q型与野生型 HbA 具有相似的氧结合能力，限制红细胞镰状化，并有可能减少血管闭塞事件 （VOE），这是一种一次性疗法。在蓝鸟生物公布的一项为期24个月、涉及12至50岁镰刀型细胞贫血病和VOE病史患者的单臂多中心研究中，表明Lyfgenia的安全性与有效性良好，该临床研究对Lyfgenia输注后6至18个月内实现VOE完全消退（VOE-CR）的患者进行了跟踪研究，结果显示，在研究的32例患者中，30位患者（94%） 的VOE得到有效治疗，其中的28位（88%）成功达到了VOE-CR，这其中包括了8例青少年患者。2、基于病毒载体的体内基因疗法（1）Gendicine/今又生公司：由深圳市赛百诺公司开发。上市时间：2003年在中国获批上市。适应症：用于治疗头颈部鳞状细胞癌。备注：重组人p53腺病毒注射液Gendicine/今又生是深圳市赛百诺公司拥有自主知识产权的腺病毒载体基因治疗药物，该药物由正常人肿瘤抑制基因p53 和人工改造的重组复制缺陷型人5型腺病毒组成，前者是药物发挥抗肿瘤作用的主体结构，后者主要起载体作用，腺病毒载体携带治疗基因p53进入靶细胞内，在靶细胞内表达抑癌基因p53，其基因表达产物可上调多种抗癌基因和下调多种癌基因活性，从而增强机体抑癌作用，实现杀伤肿瘤的目的。（2）Rigvir公司：由拉脱维亚Latima公司开发。上市时间：2004年在拉脱维亚获批上市。适应症：用于黑色素瘤治疗。备注：Rigvir是由一种基于基因修饰的ECHO-7肠道病毒载体基因疗法，目前该药已在拉脱维亚、爱沙尼亚、波兰、亚美尼亚、白俄罗斯等地被采用，同时也正在进行欧盟国家EMA的注册登记。过去十几年的临床案例证明，Rigvir溶瘤病毒安全、有效，能提高黑色素瘤患者4-6倍的生存率，此外该疗法也适用于多种其它癌症，包括结肠直肠癌、胰腺癌、膀胱癌、肾癌、前列腺癌、肺癌、子宫癌、淋巴肉瘤等。（3）Oncorine/安科瑞公司：由上海三维生物公司研发。上市时间：2005年在中国获批上市。适应症：治疗头颈部肿瘤、肝癌、胰腺癌、宫颈癌等多种癌症。备注：Oncorine (安科瑞)是一种以腺病毒为载体的溶瘤病毒基因治疗产品，该药物是利用基因工程技术删除人5型腺病毒E1B-55kD基因片段和E3-19KD基因片段后重新获得的一种溶瘤腺病毒，其在p53 基因缺乏或异常的肿瘤中能够特异性地复制，导致肿瘤细胞裂解，从而杀灭肿瘤细胞。而不损害正常细胞。临床显示安柯瑞对多种恶性肿瘤具有良好的安全性和有效性。（4）Rexin-G公司：由Epeius生物技术公司研发。上市时间：2005年获菲律宾食品与药品管理局（BFAD）批准上市。适应症：用于治疗对化疗产生抵抗的晚期癌症。备注：Rexin-G是一种载基因的纳米颗粒注射剂，通过逆转录病毒载体向靶细胞引入细胞周期蛋白G1突变基因，特异地杀死实体瘤，给药方式为静脉输注。作为主动寻找并摧毁转移性癌细胞的肿瘤靶向药物，对包括靶向生物制剂药在内的其它癌症药品无效的患者具有一定的疗效。（5）Glybera公司：由uniQure公司研发。上市时间：2012年在欧洲获批上市。适应症：治疗严格限制脂肪饮食却仍然发生严重或反复胰腺炎发作的脂蛋白脂酶缺乏症(LPLD)。备注：Glybera（alipogene tiparvovec）是一款基于AAV为载体的基因治疗药物，该疗法以AAV作为载体，将治疗基因LPL转导入肌细胞，从而使相应的细胞能够产生一定数量的脂蛋白脂酶，起到缓解疾病的作用，该疗法，一次给药长期（药效可持续多年）有效。该药物于2017年退市，其退市原因可能与定价太高、市场需求受限两大因素有关。该药物平均一次疗法费用高达100万美元，上市至今只有一位患者购买使用过，虽然医疗保险公司对其报销了90万美元，但对于保险公司而言这也是较大的负担。此外，该药物针对的适应症过于罕见，发病率约为1/100万而且误诊率较高。（6）Imlygic公司：由安进公司(Amgen)开发。上市时间：2015年先后在美国和欧盟获批上市。适应症：治疗不能经手术完全切除的黑色素瘤病灶。备注：Imlygic是一种经过基因过程技术改造的（删除其ICP34.5和ICP47基因片段，同时在病毒中插入了人粒细胞巨噬细胞集落刺激因子GM-CSF基因）减毒1型单纯疱疹病毒（HSV-1）溶瘤病毒，是首款FDA批准上市的溶瘤病毒基因疗法。给药方式为病灶局部注射（intralesional injection），将其直接注射到黑色素瘤病灶中可造成肿瘤细胞的破裂，并释放出肿瘤源性抗原和GM-CSF，促进抗肿瘤免疫应答。（7）Luxturna公司：由罗氏旗下Spark Therapeutics公司研发。上市时间：2017年获FDA批准上市，之后于2018年在欧洲获批上市。适应症：用于因双拷贝RPE65基因突变所致视力丧失但保留有足够数量的存活视网膜细胞的儿童和成人患者的治疗。备注：Luxturna是一款基于AAV的基因疗法，给药方式是视网膜下注射。该基因疗法以AAV2作为载体，将正常RPE65基因的功能性拷贝导入患者视网膜细胞，使相应细胞细胞表达正常的RPE65蛋白，弥补患者RPE65蛋白缺陷，从而改善患者视力。（8）Zolgensma公司：由诺华公司旗下AveXis公司研发。上市时间：2019年5月获FDA批准上市。适应症：治疗2岁以下脊髓性肌萎缩症（Spinal Muscular Atrophy, SMA）患者。备注：Zolgensma是一款基于AAV载体的基因疗法，该款药物是全球唯一一款获批上市的脊髓性肌萎缩症一次性治疗方案，药物的上市开启了脊髓性肌萎缩症治疗新的一页，是一项里程碑式的进展。此基因疗法用scAAV9载体经静脉输注将正常SMN1基因导入患者体内，产生正常的SMN1蛋白，从而改善运动神经元等受累细胞的功能。相比之下，治疗SMA的药物Spinraza和Evrysdi则需要长期重复给药，Spinraza每四个月通过脊柱注射给药一次，而Evrysdi是一种需每日口服药物。（9）Delytact公司：由第一三共株式会社（Daiichi Sankyo Company Limited，TYO: 4568）公司研发。上市时间：2021年6月获得日本厚生劳动省 (MHLW) 的有条件批准。适应症：用于治疗恶性胶质瘤。备注：Delytact 是全球获批的第四款溶瘤病毒基因治疗产品，也是第一个被批准用于治疗恶性胶质瘤的溶瘤病毒产品。Delytact 是一种基因工程改造的单纯疱疹病毒 1 型 (HSV-1)溶瘤病毒，由 Todo 博士及其同事开发。Delytact 是在第二代HSV-1的G207基因组中引入额外的缺失突变，增强其在癌细胞中选择性复制及对抗肿瘤免疫反应的诱导，同时保持高安全性。Delytact 是目前进行临床评估的第一个第三代溶瘤 HSV-1。Delytact 在日本的获批主要基于一项单臂 2 期临床试验，在复发性胶质母细胞瘤患者中，Delytact 达到了一年生存率的主要终点，结果显示与G207相比，Delytact表现出更强复制力和更高的抗肿瘤活性。这在乳腺癌、前列腺癌、神经鞘瘤、鼻咽癌、肝细胞癌、结肠直肠癌、恶性周围神经鞘瘤和甲状腺癌等实体瘤模型中均有效。（10）Upstaza公司：由PTC Therapeutics, Inc.（纳斯达克股票代码：PTCT）公司研发。上市时间：2022年7月获欧盟批准上市。适应症：针对芳香族 L-氨基酸脱羧酶 (AADC) 缺乏症，被批准用于治疗年龄为18 个月及以上的患者。备注：Upstaza™（eladocagene exuparvovec）是一种以2型腺相关病毒（AAV2）为载体的体内基因疗法，患者由于编码AADC酶的基因出现突变而致病，AAV2携带编码AADC酶的健康基因，以基因补偿的形式达到治疗效果，理论上一次给药长期有效，是第一个直接注入大脑的上市基因疗法，上市许可适用于所有 27 个欧盟成员国，以及冰岛、挪威和列支敦士登。（11）Roctavian公司：由BioMarin Pharmaceutical（BioMarin）公司开发。上市时间：2022年8月获欧盟批准上市；2022年11月获英国药品和保健产品管理局（MHRA）上市许可。适应症：用于治疗无FVIII 因子抑制史且AAV5抗体阴性的严重血友病A成人患者。备注：Roctavian (valoctocogene roxaparvovec)是以AAV5为载体，使用人源肝特异性启动子HLP驱动B domain删除了的人凝血因子八（FVIII）的表达。欧盟委员会批准valoctocogene roxaparvovec上市的决定是基于该药物临床开发项目的整体数据，其中，III期临床试验GENEr8-1结果显示，与入组前一年的数据相比，单次输注valoctocogene roxaparvovec后，受试者的年出血率（ABR）显著降低，重组凝血因子VIII（F8）蛋白制剂使用频率降低，或者体内血液中F8活性显著增加。在接受治疗4周后，受试者的年F8使用率和需要接受治疗的ABR分别降低了99%和84%，统计学差异显著（p<0.001）。安全性良好，没有受试者出现F8因子抑制、恶性肿瘤或血栓副作用，也没有报告与治疗相关的严重不良事件 (SAE)。（12）Hemgenix公司：由UniQure公司开发。上市时间：2022年11月获FDA批准上市。适应症：用于治疗血友病B成人患者。备注：Hemgenix是一款基于AAV5载体的基因疗法，该药物搭载有凝血因子IX（FIX）基因变体FIX-Padua，通过静脉给药，给药后该基因可在肝脏中表达FIX 凝血因子，分泌后进入血液发挥凝血功能，从而达到治疗目的，理论上一次给药长期有效。（13）Adstiladrin公司：由Ferring Pharmaceuticals开发。上市时间：2022年12月获FDA批准上市。适应症：用于治疗卡介苗（BCG）无响应的高风险非肌层浸润性膀胱癌（NMIBC）。备注：Adstiladrin是一种基于非复制型腺病毒载体的基因疗法，可以在靶细胞内过表达干扰素 alfa-2b 蛋白，给药方式是通过导尿管进入膀胱（每三个月给药一次），病毒载体可有效感染进入膀胱壁细胞，之后过表达干扰素 alfa-2b 蛋白以发挥治疗作用。这种新颖的基因治疗方法从而将患者自身的膀胱壁细胞转变为生产干扰素的微型“工厂”，从而增强患者的抗癌能力。Adstiladrin 的安全性和有效性在一项多中心临床研究中得到评估，该临床研究包括 157 名高危 BCG 无反应 NMIBC 患者。患者每三个月接受一次 Adstiladrin，持续长达 12 个月，或直到对治疗产生不可接受的毒性或复发高级别 NMIBC。总体而言，使用 Adstiladrin 疗法的入组患者中有 51% 获得了完全缓解（膀胱镜检查、活检组织和尿液中所见的所有癌症迹象均消失）。（14）Vyjuvek公司：由Krystal Biotech公司开发。上市时间：2023年5月19日获FDA批准上市。适应症：用于 6 个月及以上患有营养不良性大疱性表皮松解症 (DEB) 患者的治疗。备注：Vyjuvek是一款以基因改造后的单纯疱疹病毒为载体的基因疗法，该药物被制成凝胶剂型，给药方式为创口局部涂抹，给药频率约为每周一次。该病毒载体搭载 COL7A1 基因的正常拷贝，可将该基因递送至患者伤口部位的靶细胞内。COL7A1基因可在靶细胞内过表达COL7 蛋白，COL7 蛋白分子可自行排列成细长的束，将表皮和真皮结合在一起，这对于维持皮肤的完整性至关重要。（15）Elevidys公司：由Sarepta Therapeutics与罗氏（Roche）联合开发。上市时间：2023年6月22日获FDA批准上市。适应症：用于治疗 4-5 岁、可独立行走的杜氏肌营养不良（DMD）儿童，此外，该药品在外显子8和/或外显子9上存在缺失突变的DMD儿童中禁用。备注：Elevidys（delandistrogene moxeparvovec，SRP-9001）药物是一款基于AAV载体的基因疗法，其搭载的DNA包含一个截短型抗肌萎缩蛋白基因，该基因受MHCK7 启动子/增强子的控制，能够优先在心肌和骨骼肌中的表达。病毒血清型为恒河猴 74 型 (rAAVrh74) ，是非人类灵长类动物来源的，预期可以降低患者对载体具有预存抗体的可能性（大约 13.9‑15% 的目标人群中存在针对 AAVrh74 的抗体）。 （16）BEQVEZ公司：由辉瑞公司开发。上市时间：2024年1月3日获加拿大卫生部批准上市。适应症：用于治疗中度至重度血友病B且对AAV血清型Rh74的中和抗体检测为阴性的成年（18岁及以上）患者。备注：BEQVEZ（fidanacogene elaparvovec）是一种体内基因治疗药物，利用基因工程改造的AAV衣壳提高了将高活性人类FIX基因递送到人体目标组织器官的能力，给药方式为静脉输注，一次给药长期甚至终生有效。加拿大卫生部批准BEQVEZ基于一项开放标签、单臂的三期BENEGENE-2研究，该研究旨在评估fidanacogene elaparvovec在年龄为18至65岁的中度至重度血友病B男性参与者中的疗效和安全性，这项研究共纳入了45名参与者。临床数据显示，BENEGENE-2试验达到了预期的主要目标。相较于传统的常规治疗方案，接受BEQVEZ输注的患者在年化出血率（ABR）上展现出了良好的疗效和安全性，辉瑞此前公布的数据显示，这种基因疗法成功地将患者的ABR降低了71%，年化凝血因子输注率降低了92%。此外，接受BEQVEZ输注后，患者体内FIX活性维持稳定。3、小核酸药物（1）Vitravene公司：由Ionis Pharma（原Isis Pharma）和诺华联合开发。上市时间：1998年和1999年先后获FDA和欧盟EMA批准上市。适应症：用于治疗HIV阳性患者的巨细胞病毒性视网膜炎。备注：Vitravene是一款反义寡核苷酸药物，是全球最早获批上市的寡核苷酸药物。上市初期，市场对于抗巨细胞病毒药物的需求甚为迫切；随后由于高活性抗逆转录病毒疗法的发展，巨细胞病毒病例数量急剧下降，因市场需求低迷，该药物于2002年和2006年先后在欧盟国家和美国撤市。（2）Macugen公司：由辉瑞（Pfizer）和Eyetech共同研发。上市时间：2004年在美国获批上市。适应症：用于治疗新生血管性年龄相关性黄斑变性。备注：Macugen是一款聚乙二醇化的修饰性寡核苷酸药物，该产品能够靶向结合血管内皮生长因子（VEGF165亚型），给药方式为玻璃体内注射。（3）Defitelio公司：由爵士制药（Jazz）研发。上市时间：2013年获欧盟批准上市，2016年3获FDA批准上市。适应症：用于治疗肝小静脉闭塞症伴随造血干细胞移植后肾或肺功能障碍。备注：Defitelio是一款寡核苷酸药物，为一种具有纤溶酶特性的寡核苷酸混合物。由于商业原因于2009年撤市。（4）Kynamro公司：由Ionis Pharma和Kastle共同开发。上市时间：2013年作为孤儿药在美国获批上市。适应症：用于辅助治疗纯合子家族性高胆固醇血症。备注：Kynamro是一款反义寡核苷酸药物，是一种以人类apo B-100 mRNA为靶点的反义寡核苷酸。Kynamro的给药方式为200mg皮下注射，每周1次。（5）Spinraza公司：由Ionis Pharmaceuticals公司研发。上市时间：2016年12月获FDA批准上市。适应症：用于治疗脊髓性肌萎缩症（SMA）。备注：Spinraza （nusinersen）是一款反义寡核苷酸药物，Spinraza通过与SMN2外显子7的剪切位点结合，能够改变SMN2基因的RNA剪切，进而增加全功能性SMN蛋白的生产。2016年年8月，BIOGEN公司行使了选择权获得了Spinraza的全球权利。Spinraza在2011年才开始第一次在人体进行临床试验，短短5年时间，到2016年就获得FDA批准上市，体现了FDA对其疗效的充分认可。该药物于2019年4月在中国获批上市，Spinraza在国内的整个审批周期不到6个月，距离Spinraza在美国首次获批2年零2个月，这样一个重磅的国外罕见病新药在中国上市的速度已经算是很快了。这也得益于药品审评中心在2018年11月1日发布了《关于发布第一批临床急需境外新药名单的通知》，列入首批40个重点加速审评的国外新药，Spinraza位列其中。（6）Exondys 51公司：由AVI BioPharma公司(后改名为Sarepta Therapeutics)开发。上市时间：2016年9月获得FDA批准上市。适应症：用于治疗DMD基因发生第51号外显子跳跃基因突变的杜氏肌营养不良症（DMD）。备注：Exondys 51是一款反义寡核苷酸药物，该反义寡核苷酸可以与DMD基因的pre-mRNA 51号外显子位置结合，导致在形成成熟mRNA后，51号外显子部分被切除，从而部分修正mRNA读码框，帮助患者合成一些有一定功能形式的相对正常蛋白短一点的抗肌萎缩蛋白，从而改善患者症状。（7）Tegsedi公司：由Ionis Pharmaceuticals公司开发。上市时间：2018年7月获欧盟批准上市。适应症：用于治疗遗传性转甲状腺素蛋白淀粉样变性(hATTR)。备注：Tegsedi是一款靶向转甲状腺素蛋白mRNA的反义寡核苷酸药物，为全球首个获批治疗hATTR的药物，给药方式为皮下注射。该药物通过靶向转甲状腺素蛋白（ATTR）的mRNA，降低ATTR蛋白的产生，在治疗ATTR方面具有良好的效益风险比，患者神经病变和生活质量方面获得实质性改善，并与TTR突变类型、疾病分期或心肌病存在均无关。（8）Onpattro公司：由Alnylam公司和Sanofi公司共同开发。上市时间：2018年在美国获批上市。适应症：用于治疗遗传性转甲状腺素蛋白淀粉样变性(hATTR)。备注：Onpattro是一款靶向转甲状腺素蛋白mRNA的siRNA药物，该药物通过靶向转甲状腺素蛋白（ATTR）的mRNA，降低肝脏中ATTR蛋白的产生，减少周围神经中淀粉样沉积物的积累，进而改善和缓解疾病症状。（9）Givlaari公司：由Alnylam公司研发。上市时间：2019年11月获FDA批准上市。适应症：用于治疗成人急性肝卟啉症（acute hepatic porpyria，AHP）。备注：Givlaari是一款siRNA药物，是继Onpattro之后，第二款获批上市的siRNA药物。给药方式为皮下注射，该药物靶向降解ALAS1蛋白的mRNA，每月接受Givlaari治疗能够显著持久地降低肝脏中ALAS1的水平，从而将具有神经毒性的ALA和PBG的水平降低到正常范围，从而缓解患者的疾病症状。数据显示，与安慰剂组相比，接受Givlaari治疗的患者疾病发作次数降低74%。（10）Vyondys53公司：由Sarepta Therapeutics公司开发。上市时间：2019年12月获FDA批准上市。适应症：用于抗肌萎缩蛋白基因外显子53剪切突变的DMD患者的治疗。备注：Vyondys 53是一款反义寡核苷酸药物，该寡核苷酸药物靶向抗肌萎缩蛋白mRNA前体的剪接过程，在抗肌萎缩蛋白mRNA前体间接过程中，将其中的外显子53部分剪切掉，即不出现在成熟的mRNA上，旨在产出截短但仍具有功能的抗肌萎缩蛋白，从而改善患者运动能力。（11）Waylivra公司：由Ionis Pharmaceuticals公司及其子公司Akcea Therapeutics研发。上市时间：2019年5月获欧洲药物管理局（EMA）批准上市。适应症：作为家族性乳糜微粒血症综合征（FCS）成年患者控制饮食之外的辅助疗法。备注：Waylivra是的一款反义寡核苷酸药物，此药是全球首个获批上市用于治疗FCS的药物。（12）Leqvio公司：由诺华（Novartis）公司开发。上市时间：2020年12月获欧盟批准上市。适应症：用于治疗成人原发性高胆固醇血症（杂合子家族性和非家族性）或混合型血脂异常。备注：Leqvio是的一款靶向PCSK9 mRNA的siRNA药物，是全球首款降胆固醇（LDL-C）的siRNA疗法，给药方式为皮下注射。该药物通过RNA干扰作用，降低PCSK9蛋白水平，进而降低LDL-C水平。临床数据显示，对于使用最大耐受剂量的他汀类药物治疗后仍无法使LDL-C水平下降至达标水平的患者，Leqvio可使LDL-C下降约50%。（13）Oxlumo公司：由Alnylam制药公司开发。上市时间：2020年11月获欧盟批准上市。适应症：用于治疗原发性高草酸尿症1型（PH1）。备注：Oxlumo是一款靶向羟基酸氧化酶1（HAO1）mRNA的siRNA药物，给药方式为皮下注射。该药物采用了Alnylam公司最新的增强稳定化学ESC-GalNAc共轭技术开发，该技术可使皮下给药的siRNA具有更强的持久性和药效。该药物通过靶向降解或抑制羟基酸氧化酶1（HAO1）mRNA，降低肝脏中乙醇酸氧化酶的水平，进而消耗生成草酸所需的基质，减少草酸生成来控制患者疾病进展，改善疾病症状。（14）Viltepso公司：由日本新药株式会社（Nippon Shinyaku）子公司NS Pharma开发。上市时间：2020年8月获FDA批准上市。适应症：用于治疗DMD基因发生第53号外显子跳跃基因突变的杜氏肌营养不良症（DMD）。备注：Viltepso是一款反义寡核苷酸药物，可以与DMD基因的pre-mRNA 53号外显子位置结合，导致在形成成熟mRNA后，53号外显子部分被切除，从而部分修正mRNA读码框，帮助患者合成一些有一定功能形式的相对正常蛋白短一点的抗肌萎缩蛋白，从而改善患者症状。（15）Amondys 45公司：由Sarepta Therapeutics公司研发。上市时间：2021年2月获FDA批准上市。适应症：用于治疗DMD基因发生第45号外显子跳跃基因突变的杜氏肌营养不良症（DMD）。备注：Amondys 45是一款反义寡核苷酸药物，该反义寡核苷酸可以与DMD基因的pre-mRNA 45号外显子位置结合，导致在形成成熟mRNA后，45号外显子部分被切除，从而部分修正mRNA读码框，帮助患者合成一些有一定功能形式的相对正常蛋白短一点的抗肌萎缩蛋白，从而改善患者症状。（16）Amvuttra（vutrisiran）公司：由Alnylam制药公司开发。上市时间：2022年6月获FDA批准上市。适应症：用于治疗成人遗传性转甲状腺素蛋白淀粉样变性伴多发性神经病（hATTR-PN）。备注：Amvuttra（Vutrisiran）是一款靶向转甲状腺素蛋白（ATTR）mRNA的siRNA药物，给药方式是皮下注射。Vutrisiran基于Alnylam的增强稳定化学（ESC）-GalNAc偶联递送平台设计，增加了效力和代谢稳定性。该疗法的获批是基于其III期临床研究（ HELIOS-A ）9个月的数据，整体结果表明：该疗法改善了hATTR-PN的症状，超过50%的患者病情得到了逆转或停止恶化。（17）Qalsody（tofersen）公司：由Biogen和合作伙伴Ionis Pharmaceuticals共同开发。上市时间：2023年4月获FDA批准上市。适应症：用于治疗超氧化物歧化酶1 (SOD1)突变所致的肌萎缩侧索硬化（ALS，渐冻症）。备注：Qalsody（tofersen）是一款反义寡核苷酸药物，该反义寡核苷酸可以与与编码SOD1的mRNA结合，造成其被核糖核酸酶RNase-H降解，从而减少突变SOD1蛋白的生成。（18）Rivfloza（nedosiran）公司：该药物最初由Dicerna Pharmaceuticals研发，诺和诺德在2021年收购该公司后继续开发。上市时间：2023年10月获FDA批准上市。适应症：用于降低9岁及以上儿童和成人1型原发性高草酸尿症（PH1）患者的尿草酸水平。备注：该药物是一款靶向肝乳酸脱氢酶（LDH）mRNA的siRNA药物，给药方式是皮下注射，给药频率为每月一次。（19）Wainua（Eplontersen）公司：由Ionis Pharmaceuticals和阿斯利康联合开发。上市时间：2023年12月21日获 批准上市。适应症：用于治疗成人遗传性转甲状腺素蛋白介导的淀粉样变性的多发性神经病 。备注：该药物是一款反义寡核苷酸药物，给药方式是皮下注射，给药频率为每月一次。通过抑制TTR蛋白的产生，从而治疗淀粉样变性多发性神经病。其采用的反义寡核苷酸配体偶联（LICA）技术，可以将ASO药物与能和细胞表面特定受体结合的配体分子偶联在一起。4、其它基因治疗药物（1）Neovasculgen公司：由Human stem cells institute公司研发。上市时间：2011年12月7日在俄罗斯获批上市，此后于2013年在乌克兰上市。适应症：用于治疗周边血管动脉疾病，包括重度肢体缺血。备注：Neovasculgen是一种基于DNA质粒的基因疗法，将血管内皮生长因子（VEGF）165基因构建于质粒骨架上，输注给患者。（2）Collategene公司：由大阪大学和风投公司共同研发。上市时间：2019年8月获日本厚生劳动省批准上市。适应症：治疗重症下肢缺血。备注：Collategene是一款的基于质粒的基因疗法，此基因治疗药物AnGes公司生产的首个日本本土基因治疗药物。此药的主要成分是含人体肝细胞生长因子（HGF）基因序列的裸露质粒，若注射该药物到下肢肌肉，表达的HGF会促进闭塞的血管周围形成新的血管。临床试验证实了其改善溃疡的效果。嘉宾阵容以及授课话题已曝光，点击图片查询，合作热线：王晨 180 1628 8769戳“阅读原文”立即领取限量免费参会名额!

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        近年来，核酸类药物因为设计简单、研发周期短、成功率高、特异性强、能够在根本上调控致病基因等优势，成为众多难治疾病治疗方案的研究热点。不过核酸类药物自身也存在一些问题，如体内稳定性差，容易被体内的核酸酶降解或被肾脏清除；核酸类药物本身分子量大且带有负电荷，难以跨越细胞膜到达胞内来发挥作用；核酸类药物在体内的靶向性弱，不能精准靶向目标细胞或组织，可能会在实际应用中产生脱靶效应，导致副作用从而损伤机体，进入细胞内的药物还会面临被溶酶体水解的风险，以及在循环过程中非特异性吸附蛋白而无法发挥作用等。因此，除通过化学修饰改善核酸稳定性和免疫原性外，还需为其提供高效安全的体内递送系统。当前核酸药物开发可分为化学改造和物理改性两大方向，二者可单独或共同提升核酸药物体内外稳定性和特定组织、细胞内的翻译效率。目前已开发出多种核酸改造和递送技术，主要包括化学修饰、配体偶联和纳米递送载体。       化学修饰核酸化学修饰广泛用于ASO、siRNA、miRNA、mRNA等核酸类药物，有助于核酸类药物在疾病治疗中发挥作用。对核酸类药物进行修饰能够提高其抗酶解能力，保持序列的稳定性并延长半衰期，可以增加药物的脂溶性，降低免疫原性。同时，化学修饰可以提高靶向性，如在核酸序列的末端引入细胞表面特异性识别的配体或适配体，提高核酸类药物的沉默效率和催化反应效率等。       1、磷酸基团修饰       磷酸基团的修饰常在非桥连氧原子上进行，应用较为广泛的是硫原子取代磷酸基团的一个非桥连氧原子，从而形成硫代磷酸酯键。硫代磷酸酯键有利于提高核酸的抗酶解能力和与血浆蛋白的结合能力，从而延长其在体内的循环时间。然而，硫代修饰的不足之处在于其与靶序列的结合力较弱，且高含量的硫代磷酸酯键可能带来细胞毒性和免疫刺激等副作用。因此，硫代磷酸酯键的位置和数量对于递送效率也很重要。此外，磷酸基团上的氧原子还可被各种胺基、硼烷基取代，或整个磷酸基被酰胺基、胺氧基、烷氧基、三氮唑基等取代，但这些应用均不如硫代修饰普遍。       2、碱基修饰       对碱基的修饰主要为碱基的取代基修饰或碱基的替换，嘧啶的5-位和嘌呤的8-位是常用的取代位点。常用的碱基修饰类型有假尿苷、2-硫尿苷、N1-甲基假尿苷、5-甲基尿苷、5-甲氧基尿苷、N6-甲基腺苷、5-甲基胞苷等。其中用假尿苷代替尿嘧啶是常见的碱基修饰之一。研究证明，替换成假尿苷可改善编码Cas9 的 mRNA 的翻译并降低其免疫原性。此外，N-乙基哌啶-6-三唑修饰的腺苷类似物可以阻碍siRNA 与Toll样受体8的相互作用，降低免疫原性。6'-苯基吡 咯胞嘧啶是一种胞嘧啶类似物，具有良好的碱基配对效应、热稳定性和强荧光性。siRNA 经 6'-苯基吡 咯胞嘧啶修饰后其基因沉默效果不受影响，且其荧光特性可用于检测细胞内 siRNA 的摄取和运输。       3、核糖修饰       核糖修饰主要有两种类型，一种是在其 2' 位引入不同大小和极性的基团，常见的有 2'-甲氧基、2'-甲氧基乙氧基和 2'-脱氧-2'-氟等；另一种是在 2' 位及其他核糖位点同时进行修饰，如锁核酸 (locked nucleic acid，LNA)、肽核酸 (peptide nucleic acid，PNA) 和磷酰二胺吗啉代寡核苷酸 (phosphoramidite morpholino oligonucleotides，PMO) 等。2'位修饰对于抑制核酸酶的水解很重要。2'-甲氧基修饰是目前应用较广泛的核糖修饰手段，可以增强药物与靶 mRNA 的结合性、抑制核酸酶的水解、减弱体内免疫原性，并赋予核酸结构一定的脂溶性。2'-甲氧基乙氧基是 2'-甲氧基的类似物，具有相似的性质，但对靶 mRNA 的亲和力和抗酶解能力更强。2'-脱氧-2'-氟修饰可以提高 siRNA 的亲和力和稳定性。在核糖的2'、4'位甚至整个糖环进行修饰也会产生较好的效果。       锁核酸是一种核酸构象的限制性修饰，采用 C3'-内切构象，其可以仅用较短的序列来保持较高的靶亲和力和抗酶解能力。但短序列的锁核酸的脱靶效应和毒性问题较为严重，因此实际应用中常采用解锁核酸、限制性乙基桥核酸、三环 DNA、乙二醇核酸来替代，并且将这四种核糖修饰与硫代磷酸酯修饰组合运用可以获得更好的治疗效果。PMO和肽核酸修饰带来的电中性结构可以提高核酸类药物的稳定性和亲和力。PMO 的水溶性较好，应用较为广泛，目前一些 PMO 修饰的 siRNA 和miRNA 已进入临床试验阶段。PMO 还可以进一步修饰，如引入穿膜肽和带正电性的胺基，以提高其跨膜能力。       偶联配体核酸药物可通过共价偶联脂质、半乳糖、蛋白/多肽或适配体等来延长体内循环时间，增加在特定组织和细胞的蓄积与摄取。利用特定的偶联配体不但能调节核酸药物与血浆蛋白的结合，改善药物的组织分布，还能通过配体靶向细胞表面受体实现特定组织或细胞的递送。同时，还可通过设计配体与核酸药物之间的连接键，如采用酸敏感的酰胺键、在胞质中易被还原断开的二硫键或 Dicer 敏感键等，使核酸药物在进入特定生理环境后与偶联物分离，有效适应特定的治疗机制。       1、脂质偶联       寡核苷酸共价结合脂质及其衍生物形成的缀合物可增强寡核苷酸的体内递送效率。如在3'末端共价键合胆固醇的siRNA可有效沉默肌肉生长抑素诱导肌肉生长，共价键合α-生育酚的siRNA可沉默小鼠肝脏中ApoB。偶联胆固醇的RNA可通过与高密度脂蛋白 (high-density lipopro‐tein，HDL)或低密度脂蛋白(low-density lipoprotein，LDL) 的预组装，可增加特定组织和细胞的摄取，改变siRNA 的分布。与游离siRNA-胆固醇相比，和HDL预组装的 siRNA-胆固醇(HDL siRNA) 可在肝脏和空肠中产生更强的沉默效应。进一步研究发现，与 LDL预组装的siRNA-胆固醇(LDL siRNA)颗粒几乎只在肝脏中吸收，而HDL siRNA 颗粒主要由肝脏、肾上腺、卵巢、肾脏和小肠吸收。因此，研究者提出 siRNA-胆固醇的内吞作用分别由HDL和LDL相关的B类I型清道夫受体和LDL受体介导。还有研究发现siRNA-脂质缀合物与不同类别脂蛋白的体内结合效力受其疏水性影响，疏水性更强的缀合物优先结合LDL，而疏水性弱的缀合物优先结合HDL。因此可以通过偶联不同疏水性的脂质实现 RNA 药物在不同组织的靶向递送。       2、N-乙酰氨基半乳糖 (N-acetylgalactosamine，GalNAc) 偶联       GalNAc是一种与去唾液酸糖蛋白受体(asialoglycoprotein receptor，ASGPR) 高度亲和的二糖类化合物。ASGPR在肝脏中高度特异性表达，采用GalNAc偶联的RNA药物有望实现肝脏的高效特异性递送。2019年，第1个靶向δ-氨基乙酰丙酸合酶1的GalNAc偶联siRNA药物Givosiran 获批用于治疗急性间歇性卟啉症患者。2020年底，两种GalNAc偶联siRNA 药物lumasiran 和inclisiran 也分别获批临床使用。Lumasiran 是一种用于治疗1型原发性高草酸尿症的siRNA 药物，可降低肝细胞中羟基酸氧化酶1的mRNA的翻译。Inclisiran是一种用于治疗动脉粥样硬化性心血管疾病的 siRNA药物，可降低前蛋白转化酶枯草杆菌蛋白酶/kexin 9型的mRNA 的翻译。然而ASGPR只在高度分化的肝细胞中高表达，而在低度分化的肝癌细胞系中表达水平较低，GalNAc 偶联用于肝癌治疗的策略仍有待改进。此外，由于肝外组织缺乏ASGPR 的表达，尚无法通过GalNAc 偶联技术实现高效的 RNA 药物递送。       3、多肽偶联       细胞穿透肽 (cell penetrating peptide，CPP) 一般是由5～30个氨基酸组成的两亲性或阳离子肽片段，包括HIV-1 (human immunodeficiency virus-1)反式激活因子 (HIV-Tat)、果蝇的触角足同源异形域的DNA 结合结构域第 3 片段 (peneytatin 1)、由神经肽甘丙肽和乳突蛋白合成的嵌合肽 (transportan) 或基于碱性氨基酸 (如精氨酸和赖氨酸) 的聚合物等。CPP 与负电核酸或电中性的改造核酸偶联后，可增加偶联物整体的正电性，促进核酸的细胞摄取。当前已有多个多肽-PMO (peptide-PMO， PPMO) 缀合物处于临床前研究阶段。PPMO M23D-B能增强肌营养不良蛋白外显子 23 的跳跃，实现疾病模型小鼠肌肉中肌营养不良蛋白的持续表达，富含精氨酸的 Pip6a 肽与 PMO的缀合物 (Pip6a-PMO) 可直接携带核酸进入脊髓性肌萎缩模型动物的中枢神经系统，通过靶向CUG 重复扩增的转录本，增强运动神经元存活蛋白的表达，治疗I型强直性营养不良(myotonic dystrophy 1，DM1)。除CPP 外，靶向胰高血糖素样肽1受体 (glucagonlike peptide 1 receptor，GLP1R) 的GLP1 样肽和多价环状精甘天冬氨酸肽(cyclic arginylglycylaspartic acid，cRGD) 也已证明可介导RNA 递送。研究者将由40个氨基酸构成的GLP1样肽修饰于ASO羧基末端，可在胰岛β细胞中沉默靶基因。cRGD 修饰的siRNA可特异性递送至高表达αvβ3 整合素的黑色素瘤细胞，敲低靶基因。上述研究表明多肽偶联可改善 RNA 的组织分布和细胞摄取。不过，多肽与 RNA之间也可能因异种电荷而相互吸引而导致团聚，进而影响RNA 的递送效力。       4、抗体/适配体偶联       目前已有3种非结合型 PMO药物eteplirsen、golodirsen和viltolarsen 获批用于治疗杜氏肌营养不良症，然而，其改善肌肉的效果仍有待提高。研究者尝试通过受体介导的方法增强用于肌肉疾病的寡核苷酸药物递送，如偶联转铁蛋白受体 (trans‐ferrin receptor，TfR) 的抗体加强肌肉组织递送效率。TfR作为铁结合转铁蛋白的主要入口，在高代谢活性的骨骼肌细胞、心肌细胞、脑内皮细胞和增殖性肿瘤细胞中高表达，可在网格蛋白介导下将偶联物内化。通过此途径，TfR 能促使 siRNA-抗体/适配体偶联物进入肌肉或跨越血脑屏障进入中枢神经系统，沉默特定基因的表达。基于受体-配体的相互作用，已有多种受体被证实可提高 siRNA和ASO 的靶向递送效率，包括人类表皮生长因子受体2、T 细胞标记物 CD7、转铁蛋白受体 CD71、CD44、A 型肝配蛋白受体-2 和表皮生长因子受体等。       核酸适配体是长度为20～100 nt的单链DNA或RNA， 能形成特定且稳定的三级结构，从而与靶分子高特异性稳定结合，因此也被称为"化学抗体"。相较于蛋白抗体，核酸适配体制造简单、成本低廉、尺寸小、免疫原性低。通过将核酸适配体与特定 RNA 药物偶联，可增强目标 RNA 的体内靶向递送。前列腺特异性抗原 (prostate specific membrane antigen，PSMA) 适配体A10是第一代靶向 PSMA 阳性细胞的核酸适配体。经A10偶联的siRNA可显著抑制PSMA阳性细胞内 PLK1 和 BCL2 基因的表达，实现核纤层蛋白A/C基因沉默。类似地，gp120的RNA 适配体可通过由2'-OMe 和2'氟嘧啶修饰的富含GC 序列的核酸短链结构连接 siRNA 形成复合物，抑制模型小鼠体内的 HIV-1复制。未完待续！参考资料       [1]李丹，黄宇坤，高小玲.RNA药物递送研究进展[J].药学学报，2023，58(03)       [2]王峻峰，谭曼曼，王颖等.核酸类药物的修饰和递送研究进展[J].浙江大学学报(医学版)，2023，52(04)              作者简介：小米虫，药品质量研究工作者，长期致力于药品质量研究及药品分析方法验证工作，现就职于国内某大型药物研发公司，从事药品检验分析及分析方法验证。金笔奖征文开启，速来投稿参与！来源：CPHI制药在线声明：本文仅代表作者观点，并不代表制药在线立场。本网站内容仅出于传递更多信息之目的。如需转载，请务必注明文章来源和作者。投稿邮箱：Kelly.Xiao@imsinoexpo.com▼更多制药资讯，请关注CPHI制药在线▼点击阅读原文，进入智药研习社~