Viral Vectors in Gene Therapy Strategic Selection among Lentivirus, AAV and Beyond

Since the late 1960s, when the initial concept of "using exogenous DNA to correct genetic defects in living cells" was proposed, gene therapy has evolved from theoretical exploration to clinical trials at an exponential pace, driven by scientific progress and technological innovation. Current clinical pipelines offer hope by targeting disease roots, achieving curative breakthroughs in some highly challenging complex disorders. In May 2019, the FDA approved Zolgensma-an AAV-9-based SMN1 gene replacement therapy-for treating spinal muscular atrophy (SMA) in infants under two, the leading genetic cause of infant mortality. In December 2023, Casgevy (exagamglogene autotemcel), a lentiviral-modified CD34+ hematopoietic stem/progenitor cell (HSPC) therapy, gained FDA approval for sickle cell disease (SCD) and transfusion-dependent β-thalassemia (TDT).

The key driver behind these breakthroughs is progress in viral vectors capable of delivering therapeutic genomic payloads. Over decades, multiple vectors have been systematically evaluated in preclinical and clinical settings, with varying efficacy and safety. Among them, adeno-associated viruses (AAVs) and lentiviral vectors (LVs) have emerged as leading choices for in vivo and in vitro gene correction, respectively, due to their distinct biological properties.

AAV: From "Contaminant" to Gene Therapy Star

Adeno-associated virus (AAV) has become the most widely used in vivo gene delivery tool. Wild-type AAV was first discovered in the 1960s during electron microscopy screening of adenovirus preparations and was initially dismissed as a mere contaminant. Fortunately, curiosity-driven research later uncovered its potential, focusing on engineering it for precise therapeutic gene delivery.

The core strategy for transforming wild-type AAV into recombinant AAV (rAAV) involves replacing its native genes with a therapeutic expression cassette. Decades of optimization have yielded rAAVs retaining only the inverted terminal repeats (ITRs)-essential for replication and packaging-while the small packaging capacity (~5 kb) may limit applications for large genes.

Yet, rAAVs offer unique advantages:

1. Non-integrating genome: rAAV persists as an episome, avoiding insertional mutagenesis risks.
2. Low immunogenicity: Engineered capsids reduce innate immune responses.
3. Targeting programmability: Natural serotypes or engineered capsids enable tissue-specific delivery.

These features have driven clinical breakthroughs in:

1. Gene replacement: Compensating loss-of-function mutations (e.g., SMN1 for SMA).
2. Gene silencing: Suppressing toxic proteins via RNAi (e.g., mutant huntingtin in Huntington's disease).
3. Neurotrophic support: Delivering genes like GDNF/BDNF to slow neurodegeneration.
4. Gene editing: Delivering CRISPR-Cas9 for direct mutation repair.

Lentiviral Vectors: The Cornerstone of In Vitro Gene Editing

Lentiviral vectors (LVs) represent another major class of viral-mediated gene delivery systems. Unlike AAV, LVs serve as the preferred vector for ex vivo gene correction. However, their developmental trajectory mirrors that of AAV-progressing from discovery to becoming a core tool in gene therapy was neither deliberately planned nor without challenges.

Their precursor-vectors based on the closely related gamma retrovirus-demonstrated early promise in gene correction for severe combined immunodeficiency (SCID). A subsequent clinical trial utilized retroviral vectors to deliver the common interleukin receptor γ-chain into the bone marrow cells of 11 pediatric patients, successfully reversing the SCID phenotype. Although initially hailed as a success, the trial later revealed that some patients developed leukemia, linked to gene-insertion mutagenesis caused by the vector. These early clinical experiences, combined with the inherent limitations of retroviral vectors, including genomic instability and transduction efficiency constraints, ultimately rendered them unsuitable for overcoming technical barriers. Consequently, LVs emerged as an ideal alternative to retroviral vectors and have since evolved into the dominant method for ex vivo viral-mediated cell transduction.

Lentiviral vectors (LVs) are constructed based on the HIV-1 framework, retaining the core structural, compositional, and infectious features of the wild-type virus. Similar to HIV, LVs carry single-stranded RNA genomes that undergo reverse transcription into DNA upon cell transduction. LV-mediated gene delivery requires binding to the cell surface followed by internalization by the host cell. This infection process begins with LV attachment to the host cell, a step highly dependent on viral envelope glycoproteins. Due to the limited tropism of wild-type HIV, which hinders efficient cell transduction, recombinant lentiviral vectors (rLVs) employ pseudotyping-replacing their envelopes with those from other viruses-to enhance transduction capacity. The most widely used pseudotype is the vesicular stomatitis virus glycoprotein (VSV-G), though other viral envelopes have also been explored.

Since their emergence as a viable alternative to retroviral vectors, significant progress has been made in understanding the biological properties and unique advantages of recombinant lentiviral vectors (rLVs). Compared to their retroviral predecessors, rLVs exhibit high transduction efficiency in both quiescent and non-dividing cells. Additionally, due to their preference for safer genomic integration sites, rLVs demonstrate reduced risks of insertional mutagenesis. Leveraging these advantages, LVs have been extensively applied in monogenic disease therapies and adoptive cell therapies requiring exogenous gene delivery.

Creative Biogene: A Strategic Partner in the Era of Genomic Medicine

The growing demand for viral vectors, driven by the expanding applications of AAV-mediated in vivo gene correction and lentiviral (LV) ex vivo therapies, necessitates parallel advancements in manufacturing processes to reduce costs, enhance quality, and scale production. For instance, in AAV production, capsid-related impurities-such as those containing incomplete/incorrect genomic payloads or entirely empty capsids-are facing heightened regulatory scrutiny due to potential safety and efficacy risks. Moreover, empty and partially filled capsids not only represent lost productivity in functional virus yield but also a dual resource waste: these defective capsids can bind to and transduce host cells yet fail to deliver therapeutic benefits. Thus, process development must prioritize minimizing such impurities.

At the strategic level, viral vector manufacturing requires tools and workflows that balance cost-effectiveness with end-to-end scalability. Achieving sustainable costs hinges on rigorous screening of critical raw materials, including plasmid DNA (pDNA), cell lines, and transfection reagents. For example, targeted optimization of pDNA design and transfection reagent combinations can significantly reduce empty and partially filled AAV capsids, thereby improving overall process yield and material efficiency. Implementing orthogonal experimental optimization during preclinical process development saves time and costs in later stages, as optimized workflows can be directly scaled.

Creative Biogene empowers next-generation genomic therapy developers through its cutting-edge viral platform:

• Expertise Sharing: Delivers comprehensive knowledge and technical support for gene therapy development.
• Advanced Production Systems: Offers cost-efficient AAV/LV production systems with three key advantages:

1. Seamless transition from preclinical to clinical manufacturing
2. Streamlined process development
3. 25% lower plasmid costs compared to traditional PEI transfection, coupled with higher yields

• Scalable Manufacturing: Leverages a full-spectrum, scalable product portfolio to ensure clinical success in genomic medicine.

By integrating innovation with practicality, Creative Biogene stands as a trusted partner in translating genomic breakthroughs into viable therapies.