Viral Vector Engineering

I often see retroviral and lentiviral vectors bundled together (as below) for simplicity, but they have major differences that I wanted to address as a reminder to colleagues: 👉 Firstly, lentiviral vectors (derived from lentiviruses) are still in fact retroviruses. Therefore, a more accurate distinction would be gammaretroviral vs lentiviral vectors. The so-called 'retroviral vectors' used in the field (and used in approved CAR-T products such as Yescarta and Tecartus) are derived from Mo-MLV (Moloney murine leukemia virus) which are classified as gammaretroviruses, a genus of the family Retroviridae. Lentiviral vectors (used in approved CAR-T products such as Kymriah and Breyanzi) are derived from HIV-1 which are classified as lentiviruses, also a genus of the family Retroviridae. Also, a reminder that 'retro' refers to their ability to transcribe RNA 'back' into DNA. 👉 One major consideration between using gammaretroviral vectors (gRV) and lentiviral vectors (LV) is the promoter that will drive your gene of interest. gRV have intact LTRs (long terminal repeats) which are the natural promoter of the virus it is derived from, therefore expression of your gene of interest will be driven by the gRV LTR promoter and will be susceptible to the properties of this LTR. Upon insertion, the genomic site of integration may also be susceptible to the properties of this LTR, therefore design strategy (such as enhancer activity of the viral LTR) may need to be considered. 👉 LV, on the other hand, utilize a self-inactivating LTR design (at least in the 3rd generation vectors) therefore your gene of interest would be driven by your promoter of choice, such as a cellular EF1alpha-derived promoter. Once again, upon insertion, the genomic site of integration may also be susceptible to the properties of your promoter of choice (but not the LV LTRs), therefore design strategy may need to be considered for integration site safety (such as minimizing unwanted enhancer activity of your promoter of choice). 👉Another major molecular distinction between gRV vs LV is that the latter uses an accessory protein Rev. Rev binds a secondary RNA structure on the transfer plasmid RNA called Rev Response Element (RRE), thus allowing its export from the nucleus in the producer cells so that your LV will package the transfer RNA during production (which also contains your gene of interest). Rev is necessary for LV because the transfer RNA contains RNA stem loop structures (part of the psi packaging sequence) that contain cis-acting repressive sequences (CRS) that cause the transfer RNA to be retained in the nucleus, in the absence of Rev. This is why LV has the additional Rev plasmid. 👉You can make a lentiviral vector system that is independent of Rev (we made one such system, described here: https://lnkd.in/gZ9XQqGm) but these systems haven't become popular. 👉Image: Addgene.

𝗛𝗶𝗱𝗱𝗲𝗻 𝗖𝗿𝗶𝘀𝗶𝘀 𝗶𝗻 𝗚𝗲𝗻𝗲 𝗧𝗵𝗲𝗿𝗮𝗽𝘆: 𝟵𝟳% 𝗼𝗳 𝗟𝗲𝗻𝘁𝗶𝘃𝗶𝗿𝗮𝗹 𝗩𝗲𝗰𝘁𝗼𝗿𝘀 𝗟𝗼𝘀𝘁 𝗶𝗻 𝗣𝗿𝗼𝗱𝘂𝗰𝘁𝗶𝗼𝗻 🚨 𝗕𝗿𝗲𝗮𝗸𝗶𝗻𝗴 𝗿𝗲𝘃𝗲𝗹𝗮𝘁𝗶𝗼𝗻 𝗶𝗻 𝗴𝗲𝗻𝗲 𝘁𝗵𝗲𝗿𝗮𝗽𝘆 𝗺𝗮𝗻𝘂𝗳𝗮𝗰𝘁𝘂𝗿𝗶𝗻𝗴: Klimpel et al. (2025) are not the first to expose a devastating bottleneck but they are the latest — 87-97% of lentiviral vectors are lost during production due to retro-transduction, where producer cells cannibalize their own viral output. This isn't just a manufacturing hiccup; it's sabotaging the promise of affordable gene therapies. 𝗧𝗵𝗲 𝗥𝗲𝘁𝗿𝗼-𝗧𝗿𝗮𝗻𝘀𝗱𝘂𝗰𝘁𝗶𝗼𝗻 𝗖𝗿𝗶𝘀𝗶𝘀 For decades, we believed producer cells were immune to superinfection. That dogma crumbled when researchers discovered VSV-G pseudotyped vectors exploit ubiquitous LDLR receptors, enabling massive self-transduction. The authors of the paper show producer cells accumulate up to 469 vector copies each, decimating yields and inflating costs that keep life-saving therapies from patients who need them most. 𝗚𝗮𝗺𝗲-𝗖𝗵𝗮𝗻𝗴𝗶𝗻𝗴 𝗦𝗼𝗹𝘂𝘁𝗶𝗼𝗻𝘀 𝗼𝗻 𝘁𝗵𝗲 𝗛𝗼𝗿𝗶𝘇𝗼𝗻 There may be multiple breakthrough approaches: 🔬 𝗖𝗥𝗜𝗦𝗣𝗥-𝗘𝗻𝗵𝗮𝗻𝗰𝗲𝗱 𝗣𝗿𝗼𝗱𝘂𝗰𝗲𝗿 𝗖𝗲𝗹𝗹𝘀: The CHEDAR platform combines OAS1, PKR, and LDLR knockouts, achieving 7-fold titer increases. Next-gen triple knockouts (GBP3, BPIFC, LDAH) push improvements to 8.33-fold. 🧬 𝗧𝗿𝗮𝗻𝘀𝗰𝗿𝗶𝗽𝘁𝗶𝗼𝗻𝗮𝗹 𝗦𝘂𝗽𝗲𝗿𝗰𝗵𝗮𝗿𝗴𝗶𝗻𝗴: SPT4/SPT5 overexpression tackles premature transcription termination, yielding 11-fold improvements when combined with optimized cell lines. 🎯 𝗦𝗺𝗮𝗿𝘁 𝗘𝗻𝘃𝗲𝗹𝗼𝗽𝗲 𝗘𝗻𝗴𝗶𝗻𝗲𝗲𝗿𝗶𝗻𝗴: Alternative glycoproteins like measles virus, BaEV, or engineered VSV-G variants sidestep LDLR-mediated retro-transduction while maintaining therapeutic targeting. ⚗️ 𝗡𝗼𝘃𝗲𝗹 𝗕𝗹𝗼𝗰𝗸𝗶𝗻𝗴 𝗦𝘁𝗿𝗮𝘁𝗲𝗴𝗶𝗲𝘀: ENV-Y fusion proteins and pH modulation (6.7-6.8) offer elegant solutions to neutralize self-transduction without genetic modifications. 𝗧𝗵𝗲 𝗠𝗮𝗻𝘂𝗳𝗮𝗰𝘁𝘂𝗿𝗶𝗻𝗴 𝗥𝗲𝘃𝗼𝗹𝘂𝘁𝗶𝗼𝗻 𝗔𝗵𝗲𝗮𝗱 With the lentiviral vector market racing toward billions in value, solving retro-transduction isn't just technical optimization—it's democratizing access to gene therapies. The convergence of CRISPR cell engineering, bioprocess innovation, and envelope diversification promises to transform a 97% loss crisis into manufacturing excellence. The question isn't whether we can solve this bottleneck, but how quickly we can scale these solutions to bring gene therapies within reach of millions awaiting treatment. 𝗪𝗵𝗮𝘁 𝘀𝘁𝗿𝗮𝘁𝗲𝗴𝗶𝗲𝘀 𝗱𝗼 𝘆𝗼𝘂 𝘁𝗵𝗶𝗻𝗸 𝗵𝗼𝗹𝗱 𝘁𝗵𝗲 𝗺𝗼𝘀𝘁 𝗽𝗿𝗼𝗺𝗶𝘀𝗲 𝗳𝗼𝗿 𝗼𝘃𝗲𝗿𝗰𝗼𝗺𝗶𝗻𝗴 𝘁𝗵𝗶𝘀 𝗯𝗼𝘁𝘁𝗹𝗲𝗻𝗲𝗰𝗸 𝗶𝗻 𝗟𝗩 𝗽𝗿𝗼𝗱𝘂𝗰𝘁𝗶𝗼𝗻? 𝗟𝗲𝘁’𝘀 𝗱𝗶𝘀𝗰𝘂𝘀𝘀 𝘆𝗼𝘂𝗿 𝗶𝗻𝘀𝗶𝗴𝗵𝘁𝘀 𝗮𝗻𝗱 𝗲𝘅𝗽𝗲𝗿𝗶𝗲𝗻𝗰𝗲𝘀 𝗯𝗲𝗹𝗼𝘄! #GeneTherapy #LentiviralVectors #CGTManufacturing #Bioprocessing #TimeIsLife

This newsletter explores engineered viral vectors with reduced immunogenicity and enhanced targeting, highlighting how next-generation viral vectors are improving the safety and precision of gene therapy. Traditional AAV and lentiviral vectors face challenges such as immune recognition and off-target effects, but advances in AI-driven capsid engineering and genetic modification are creating stealth viral vectors that can evade neutralizing antibodies, enabling repeated dosing and prolonged therapeutic effects. In addition, tissue-specific viral vectors are being optimized for brain, liver, and tumor targeting, enabling high-precision gene delivery for neurodegenerative diseases and cancer treatments. Emerging hybrid viral nanoparticle systems are further enhancing immune evasion and controlled gene expression. As engineered viral vectors continue to advance, they are driving the next era of gene therapy, making treatments safer, more effective, and more widely available. Join us to stay informed about how these breakthroughs are shaping precision medicine! #GeneTherapy #ViralVectors #AAV #Lentivirus #CapsidEngineering #SyntheticBiology #PrecisionMedicine #BiotechInnovation #Immunotherapy #GeneticMedicine #CSTEAMBiotech

Concern is frequently aired about LV safety This is my final post examining these worries Part 3: Beyond CAR-T 👇 In my last two posts I explored a history of LVV safety and the context within the leading LV application – ex vivo CAR-T (see links in comments). My final post in this series will explore how in vivo applications manage safety concerns and hopefully provide you with some food for thought when designing your next LVV. 𝗣𝗮𝗿𝘁 3 – 𝗕𝗲𝘆𝗼𝗻𝗱 𝗖𝗔𝗥-𝗧 Surely the most successful application of viral vectors is CAR-T with over 34,000 patients treated. CAR-T is incredibly safe with T-cell malignancy rates lower than conventional therapies. However, ex vivo transduction of purified T-cells mitigates issues that would otherwise impact a more systemic administration: 1. Off-target transduction is mitigated due to a lack of other cell types 2. Transduction efficiency is driven by increasing vector and T-cell proximity. 3. Post transduction T-cells can be monitored for killing efficiency and any abnormal growth prior to reinfusion. To design an LVV for an in vivo CAR-T, or indeed any in vivo application, the following elements should be considered critical: ⭐ 𝗧𝗶𝘀𝘀𝘂𝗲 𝘀𝗽𝗲𝗰𝗶𝗳𝗶𝗰 𝘁𝗿𝗮𝗻𝘀𝗴𝗲𝗻𝗲 𝗽𝗿𝗼𝗺𝗼𝘁𝗲𝗿. I.e. off-target transduction will not express the transgene. Clearly this is easier for distant tissue types, but is a bit more tricky for more closely related lymphocytes - expect some leakiness. Synthetic or designed tissue specific promoters are probably the best way to manage this.  An example here is Chromatin Bioscience, which is a leading design house with a number of novel synthetic promoters incorporated into clinical assets. ⭐ 𝗣𝘀𝗲𝘂𝗱𝗼𝘁𝘆𝗽𝗲 𝘁𝗵𝗮𝘁 𝘁𝗮𝗿𝗴𝗲𝘁𝘀 𝘁𝗵𝗲 𝘁𝗵𝗲𝗿𝗮𝗽𝗲𝘂𝘁𝗶𝗰 𝗰𝗲𝗹𝗹𝘀. Apart from the safety implication here, it is clearly advantageous to minimise off-target transduction as this will significantly reduce dose requirements. My view is that large part of what AZ bought into when purchasing EsoBiotec was their differentiating targeting technology. Here is an example of why these are critical: B-cells resist CD-19 loss in malignancy (epitope escape), making CD-19 a great target! However, CAR presence in B-cells shows potential epitope escape without losing CD-19 expression My final recommendation sounds obvious, manufacture as high a quality a lentivirus as possible. 𝗗𝘂𝗵! A compressed development timeline and budget will fight against this. So the easiest win is to look to minimise vector genome splicing. Splicing can cause you issues with unwanted payload expression, packaging of incomplete genomes and variable titres. Fortunately, splice sites can be predicted with some confidence using online tools – SpliceAI should definitely be your friend. Please let me know if you enjoyed this series in the comments below. I am also keen to get your recommendations on LVV design for safety and why they are important to you!