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Lentiviral Vectors</span></div></div></header><div class="site-inner"><div class="content-sidebar-wrap"><main class="content"><article class="post-3273 post type-post status-publish format-standard has-post-thumbnail category-biotech category-cell-gene-therapy category-connect-to-science category-general entry" aria-label="AAV Vs. Lentiviral Vectors"><header class="entry-header"><h1 class="entry-title">AAV Vs. Lentiviral Vectors</h1> <span class="post-info-author"> By <a href="https://www.thermofisher.com/blog/author/lifeinthelabstaff/" title="Posts by Life in the Lab Staff" rel="author">Life in the Lab Staff</a> </span> <span class="post-info-date"> 09.27.2024 </span> </header><div class="entry-content"><p><img decoding="async" class="aligncenter size-large wp-image-3365" src="https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/09/Child_Top-Pod-4-1024x576.jpg" alt="" width="760" height="428" srcset="https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/09/Child_Top-Pod-4-1024x576.jpg 1024w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/09/Child_Top-Pod-4-300x169.jpg 300w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/09/Child_Top-Pod-4-768x432.jpg 768w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/09/Child_Top-Pod-4.jpg 1280w" sizes="(max-width: 760px) 100vw, 760px" /></p> <p> </p> <h2>Exploring Viral Vectors: Navigating Gene Therapy Options with Lentiviral, AAV, and Beyond</h2> <p> </p> <h3><strong>The Era of Genomic Medicine Has Arrived</strong></h3> <p>Steady progress and scientific innovation have moved gene therapy from the inception of an idea that foreign DNA could be used to correct a genetic deficiency in living cells in the late 1960s to the logarithmic expansion of gene therapy clinical trials that we see today. Today’s clinical pipeline offers patients hope through the intent of addressing the underlying cause, and for the first time, curing some of the most challenging and complex disease states with some impressive wins thus far.<sup>1–3</sup> In May of 2019, the FDA approved Zolgensma, an AAV-9 based SMN1 gene replacement therapy for all children under two with spinal muscular atrophy, the number one genetic cause of infant death.<sup>4</sup> Casgevy (exagamglogene autotemcel), a lentiviral-corrected CD34+ human hematopoietic stem and progenitor cell product for the treatment of sickle cell disease and transfusion-dependent b-Thalassemia, was approved by the FDA in December of 2023.<sup>5</sup> These landmark approvals have only fueled the continued enthusiasm for gene therapy-based therapeutics. Currently the number of clinical trials involving gene therapies has skyrocketed prompting the acknowledgement from scientists and clinicians alike that the era of genomic medicine has begun.</p> <p>At the heart of this unprecedented clinical progress is the foundational understanding of the vectors utilized to deliver the required corrective therapeutic genomic payload. Through the years, multiple virus types have been evaluated both in preclinical models and in clinical trials with varying degrees of success. Two vector types, adeno-associated (AAV) and lentiviral vectors (LV), have emerged as the popular virus types for <em>in vivo</em> and <em>in vitro</em> gene correction.<sup>6</sup> But what are the characteristics that make these viruses ideal for different clinically relevant applications? What are their strengths and weaknesses? Here, some of the defining characteristics of AAV and LV vectors are compared with the intent of highlighting ideal applications for each.</p> <h3><strong>Adeno-Associated Virus: From Contaminant to Gene Therapy Super Star</strong></h3> <p>AAV vectors have emerged as the most popular vector for therapeutic <em>in vivo</em> gene delivery. The wild-type virus was first discovered in the mid 1960s by electron microscopy-based screening of adenovirus preparations and was initially identified as a contaminant. <sup>7</sup> Fortunately, subsequent work done purely for the sake of scientific curiosity began to uncover the significance of this accidental discovery and further efforts focused on how to modify and utilize this vector for the delivery of genetic payloads. Repurposing of the wild-type virus for recombinant vector applications was centered around the replacement of the wild-type AAV protein-coding sequences with the therapeutic gene expression cassette. <sup>8</sup> The result of this extensive engineering effort over the years produced a vector with only the inverted terminal repeats (ITRs) remaining of the wild-type virus genome.<sup>9 </sup>Though the minimal genetic elements of the original genome exist, recombinant AAVs (rAAV) can only accommodate about a 5 kb payload, which can be limiting in some cases.<sup>10</sup></p> <p>Regardless, rAAV has several inherent advantages for clinical use. Primarily, when host cell transduction occurs, the rAAV genome does not integrate and thus there is no risk of insertional mutagenesis and low potential for immunogenicity.<sup>11</sup> Further, rAAV offers the limitless potential of targeting different cell types and tissues by specific serotypes and custom capsid designs, a feature that will prove indispensable as this technology continues to clinically mature. To date, the flexibility in AAV-targeting combined with a strong safety profile has resulted in a wide range of clinical applications for this vector. In a classical sense, AAV have been developed to replace faulty genes to compensate for loss of function mutations.<sup>12,13 </sup>However, additional therapeutic strategies deploying AAV include gene silencing to eliminate function toxicity, such as in Huntington’s Disease, have been utilized.<sup>14 </sup>Further, AAV have been used to supply genes to significantly delay disease progression, such as with neurotrophic genes in neurodegenerative syndromes.<sup>15–17 </sup>Finally, AAV have been used to deliver gene editing systems, such as CRISPR, to directly repair or edit pathogenic mutations.<sup>8</sup></p> <h3><strong>Lentiviral Vectors, A Staple in <em>Ex Vivo</em> Gene Editing</strong></h3> <p>Lentiviral vectors (LVs) represent the other major virus-mediated gene delivery system (Table 1). Unlike their AAV counterparts, LVs are the preferred vector for <em>ex vivo</em> gene correction.<sup>18</sup> However LVs share similar experiences to AAV in that the journey from their discovery to the forefront of gene therapy was not deliberate or straightforward.</p> <p>Their predecessor, vectors based on the closely related gamma retrovirus, exhibited early promise in the correction of severe combined immunodeficiency (SCID). Subsequent clinical studies utilizing the retroviral-based introduction of the common interleukin receptor γ-chain in bone marrow in 11 children was effective in correcting the SCID phenotype.<sup>19</sup> Though the trial was initially deemed a success, sadly some of these patients went on to develop leukemia that was later determined to be related to the gene-insertion mutagenesis of the vector. <sup>19 </sup>These early clinical experiences along with other stability and transduction limitations proved insurmountable for retroviral vectors. Thus, LVs emerged as an attractive alternative to retroviral vectors and over time have become the primary method of <em>ex vivo</em> viral-mediated transduction of cells.</p> <div id="attachment_3366" style="width: 739px" class="wp-caption aligncenter"><img decoding="async" aria-describedby="caption-attachment-3366" class="wp-image-3366 size-full" src="https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/09/AAV-v-LV.png" alt="Comparison table of AAV and LV vectors" width="729" height="346" srcset="https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/09/AAV-v-LV.png 729w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/09/AAV-v-LV-300x142.png 300w" sizes="(max-width: 729px) 100vw, 729px" /><p id="caption-attachment-3366" class="wp-caption-text">Table 1. Comparison of AAV and LV vectors</p></div> <p>Structurally, LVs are based on HIV-1 and have common features with the wild-type virus in terms of the overall size, composition, and function of the virus.<sup>20 </sup>Like HIV, LVs contain an RNA genome that is retrotranscribed to DNA once inside the transduced cell. LV-based gene delivery requires cell surface binding and subsequent internalization to the host cell. This infection cycle is initiated by LV binding to the host cell, a process that is heavily dependent on the viral envelope glycoproteins. Since wild-type HIV has limited tropism and results in poor cell transduction, recombinant LVs (rLVs) are pseudo-typed by substituting the envelope of other viruses to enhance their transduction potential. The most popular pseudotype is vesicular stomatitis virus (VSV G), though others have been explored. <sup>21</sup></p> <p>Our understanding of rLV biology as well as the unique benefits they possess has significantly progressed since they emerged as viable alternatives to retroviral-based vectors. Unlike their retroviral-based predecessors, rLVs can efficiently transduce resting or nondividing cells.<sup>22 </sup>Further, they offer a higher level of safety and a reduced risk of insertion mutagenesis compared to their retrovirus predecessors due to their preferential loci of integration. As a result of these benefits, LVs have been extensively used in the treatment of monogenic diseases and adoptive cell therapy trials that require exogenous gene delivery.<sup>23 </sup>Further, LVs have also been extensively used in research applications for gene editing and modulation of gene expression.<sup>24</sup></p> <h3><strong>Thermo Fisher Scientific is a Dedicated Partner in the Age of Genomic Medicine </strong></h3> <p>As the use cases for either <em>in vivo</em> gene correction by AAV or the <em>ex vitro</em> use of LV continues to drive the demand for these vectors, improvements in manufacturing to reduce vector cost while enhancing quality and scale must also occur. For instance, in AAV manufacturing, product-related impurities such as capsids that contain incomplete/incorrect genomic payloads or no genomic payload at all have come under increasing scrutiny due to potential safety and efficacy concerns.<sup>25 </sup>Further, these empty and partially loaded capsids in a way represent lost productivity in the context of fully loaded and functional virus since they will bind and transduce a host cell but not be able to deliver a meaningful therapeutic response. As a result, this specific impurity as well as others must be minimized through process development.</p> <p>At a tactical level, the tools and processes to produce these vectors must be economically viable as well as scalable from end-to-end. A key driver of this cost sustainability is the careful selection of raw materials, such as pDNA, cell lines, and transfection reagents. For example, empty and partially filled AAV capsids can be minimized by targeted optimization of pDNA and transfection reagents to favorably affect the overall process yield and raw material efficiencies. Paying attention to materials optimization during the preclinical process development stages can pay off in saved time and money later, as efficient processes carry over into the manufacturing stage.</p> <p>Thermo Fisher Scientific helps support developers of these novel genomic therapeutics through multiple avenues and availability of resources. Thermo Fisher possesses extensive knowledge and expertise in <a href="https://www.thermofisher.com/us/en/home/clinical/cell-gene-therapy/gene-therapy.html">gene therapy development</a>, that can be accessed through either resources or easily available technical support. Further, Thermo Fisher provides cost-effective and industry leading <a href="https://www.thermofisher.com/us/en/home/clinical/cell-gene-therapy/gene-therapy/aav-production-workflow/adeno-associated-virus-production-research.html?ef_id=CjwKCAjw2dG1BhB4EiwA998cqJKA7l7r-bjRz7bpBmivL4RS-cP2GveshVmJ9lLkDqCf-G3ShlsaOxoCem8QAvD_BwE:G:s&s_kwcid=AL!3652!3!545957491095!e!!g!!aav%20max%20adeno-associated%20viral%20production%20system!1646441759!130225927194&cid=bid_clb_cts_r01_co_cp0000_pjt0000_bid00000_0se_gaw_nt_pur_con&gad_source=1&gbraid=0AAAAADxi_GQ57ZY8ptCI4E_8q1EBR38pv&gclid=CjwKCAjw2dG1BhB4EiwA998cqJKA7l7r-bjRz7bpBmivL4RS-cP2GveshVmJ9lLkDqCf-G3ShlsaOxoCem8QAvD_BwE">AAV</a> and <a href="https://www.thermofisher.com/order/catalog/product/A35684?SID=srch-srp-A35684">LV</a> production systems. These systems ease the transition from preclinical to clinical manufacturing, streamline process development, and cut plasmid cost by 25% with superior yields over PEI reagents. With extensive knowledge and a product portfolio committed to enhancing the scalability and efficiency of viral vector manufacturing, Thermo Fisher is committed to ensuring the clinical success of genomic medicines.</p> <h3><a href="https://www.thermofisher.com/us/en/home/clinical/cell-gene-therapy/gene-therapy.html">Explore gene therapy development solutions</a> >></h3> <p> </p> <h4><em>Want to read more stories like this? </em><a href="https://www.thermofisher.com/us/en/home/global/forms/connect-science-subscribe.html?icid=bid_cbu_sbu_r01_co_cp1605_pjt10933_blg50592_0so_blg_il_awa_og_s00_C2SOncoBlogC2SSub" target="_blank" rel="noopener"><em>Subscribe to Connect to Science</em></a><em>, your portal for life science news.</em></h4> <p> </p> <p>##</p> <p><em>For Research Use Only. Not for use in diagnostic procedures.</em></p> <p><em>© 2024 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.</em></p> <p> </p> <h3><strong>References</strong></h3> <ol> <li>Labbé, R. P., Vessillier, S. & Rafiq, Q. A. Lentiviral vectors for t cell engineering: Clinical applications, bioprocessing and future perspectives. <em>Viruses</em> vol. 13 Preprint at https://doi.org/10.3390/v13081528 (2021).</li> <li>Issa, S. S., Shaimardanova, A. A., Solovyeva, V. V. & Rizvanov, A. A. Various AAV Serotypes and Their Applications in Gene Therapy: An Overview. <em>Cells</em> vol. 12 Preprint at https://doi.org/10.3390/cells12050785 (2023).</li> <li>Kang, L. <em>et al.</em> AAV vectors applied to the treatment of CNS disorders: Clinical status and challenges. <em>Journal of Controlled Release</em> vol. 355 458–473 Preprint at https://doi.org/10.1016/j.jconrel.2023.01.067 (2023).</li> <li>Ogbonmide, T. <em>et al.</em> Gene Therapy for Spinal Muscular Atrophy (SMA): A Review of Current Challenges and Safety Considerations for Onasemnogene Abeparvovec (Zolgensma). <em>Cureus</em> (2023) doi:10.7759/cureus.36197.</li> <li>Parums, D. V. 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Therapeutic modulation of gene expression in the disease state: Treatment strategies and approaches for the development of next-generation of the epigenetic drugs. <em>Frontiers in Bioengineering and Biotechnology</em> vol. 10 Preprint at https://doi.org/10.3389/fbioe.2022.1035543 (2022).</li> <li>McColl-Carboni, A. <em>et al.</em> Analytical characterization of full, intermediate, and empty AAV capsids. <em>Gene Ther</em> <strong>31</strong>, 285–294 (2024).</li> </ol> <p> </p> <aside class="mashsb-container mashsb-main "><div class="mashsb_above_buttons">Share this article </div><div class="mashsb-box"><div class="mashsb-count mash-large" style="float:left"><div class="counts mashsbcount">217</div><span class="mashsb-sharetext">SHARES</span></div><div class="mashsb-buttons"><a class="mashicon-facebook mash-large mashsb-noshadow" href="https://www.facebook.com/sharer.php?u=https%3A%2F%2Fwww.thermofisher.com%2Fblog%2Flife-in-the-lab%2Faav-vs-lv%2F" target="_top" rel="nofollow"><span 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