The Second Wave of Viral Vectors: Where Are AAV, Lentivirus, and Adenovirus Today?

Although viruses have not yet been fully conquered, human exploration of their applications began long ago. One of the world’s most dangerous biological entities has been transformed in the laboratory into a vehicle for delivering DNA.

To date, viral vectors have undergone continuous design optimization and are now fully capable of carrying the hope for curing diseases. However, based on currently disclosed information, many challenges remain to be addressed.

Drug delivery is not a new topic. In the era of small-molecule drugs, delivery was achieved through dosage forms. Different dosage forms determine when and where small-molecule drugs are released, thereby dictating the drug’s dosage, route of administration, and mode of delivery.

The stable physicochemical properties of small-molecule drugs make them less prone to rapid degradation after entering the body, allowing them to easily reach their sites of action; therefore, they do not require complex delivery technologies for assistance. In the era of biopharmaceuticals, however, while the design of the “active ingredients” in biologics more closely mimics the body’s innate biological processes, it has become increasingly challenging to safely deliver these “active ingredients” to their target sites.

Viral vectors are among the earliest biological drug delivery technologies to be developed. The emergence of viral vectors was primarily aimed at addressing the challenge of DNA delivery. Since the latter half of the 20th century, research institutions worldwide have been attempting to use viral vectors to encapsulate genetic information for the curative treatment of genetic disorders. This series of bold endeavors reached its peak in the 1990s, marked by positive outcomes from a clinical study.

In 1990, Dr. William French Anderson of the National Institutes of Health (NIH) led a long-term clinical trial in which the normal adenosine deaminase (ADA) gene was delivered via retroviral vectors into T cells isolated from patients, which were then reinfused into the patients for therapeutic purposes.Adenosine Deaminase Deficiency Severe Combined Immunodeficiency (ADA-SCID)children.This clinical study observed significant improvement in children's symptoms, leading William French Anderson to be hailed as the "Father of Gene Therapy."Encouraged by this clinical study, a large number of gene therapy-related clinical trials emerged in the 1990s, particularly focusing on ADA-SCID.

Although the therapeutic efficacy was evident, some overly aggressive approaches failed to sustain the first wave of gene therapy.

In 1999, a serious clinical research accident instantly plunged this field into its lowest point.Jesse Gelsinger, an 18-year-old patient with ornithine transcarbamylase (OTC) deficiency, died in a gene therapy clinical trial. In this trial, researchers attempted to deliver the missing gene into Jesse’s body using an adenovirus vector; however, the adenovirus triggered a severe immune response, ultimately leading to Jesse’s death from multiple organ failure four days after treatment. Subsequently, the FDA shut down numerous gene therapy-related studies, and this event directly marked the end of the first wave of gene therapy.

Looking back at the first wave, this early exploration of gene therapy succeeded and failed by viral vectors.The series of issues exposed by viral vectors in this early exploration has pointed the way for subsequent research on viral vectors.

Retroviruses achieved tremendous success in this wave, but also demonstrated the dangers of random insertion for the first time.The fundamental mechanism of retroviruses involves the random integration of target DNA fragments into the host cell’s genome. The safety risks associated with this random insertion were not evident during T-cell infection studies. However, as research advanced to the more complex stage of stem cells, these safety concerns were rapidly amplified.

In 2003, five patients with ADA-SCID who had undergone gene therapy were diagnosed with leukemia. The retroviral vectors used in their treatment were the same as those employed by William French Anderson—namely, Moloney murine leukemia virus vectors. However, a key difference was that William Anderson modified and reinfused only T cells, whereas these five patients received genetically modified hematopoietic stem cell transplants. Although the patients’ immune functions showed significant recovery after treatment, they developed leukemia due to abnormal activation of the LMO2 oncogene caused by random insertion of the retroviral vector.

Subsequently, although researchers further engineered the Moloney murine leukemia virus vector, safety concerns could never be fully eliminated. To this day, the risk of insertional mutagenesis continues to plague lentiviruses, the descendants of retroviral vectors.

On the other hand, adenoviruses, once a dominant force for a decade, were replaced by adeno-associated viruses (AAVs) due to their strong immunogenicity and the potential loss of introduced circular DNA during cell division. However, with continuous development and optimization, adenoviruses with controlled immunogenicity have found new applications in the vaccine field.

Two decades after the first wave, viral vectors have once again become a hot topic of discussion with the rapid rise of the cell and gene therapy industry. Currently, virus-based delivery technologies applied in the biopharmaceutical field are primarily focused on three areas: lentiviruses, adenoviruses, and adeno-associated viruses.

Lentiviral vectors are currently the most mature viral vector type for industrial applications, playing a pivotal role in immune cell therapies, such as CAR-T therapy.

Lentiviruses are essentially a subclass of retroviruses, named for their characteristically long incubation period.Compared with other types of retroviruses, lentiviruses can penetrate the nuclear membrane, infect a wider range of cell stages, and achieve efficient infection of both dividing and non-dividing cells.In contrast, most retroviruses lack the ability to penetrate the nuclear envelope and can only enter the nucleus during mitosis. Consequently, lentiviral vectors have gradually replaced the original retroviral vector systems in development.

Currently, commonly used lentiviral systems still integrate the target fragment into the genome of infected cells through a reverse transcription process.During viral production, the plasmid expressing the capsid and the expression plasmid carrying the target fragment are completely separated. The packaged viruses do not contain the genetic sequence for the viral capsid, thereby losing their ability to replicate. Therefore, the commonly used three-plasmid system already offers a high level of safety, while the subsequently optimized four-plasmid system provides even greater safety.

Overall, the lentiviral infection system fully retains the advantages of high expression efficiency and prolonged expression duration characteristic of original retroviruses, while demonstrating a substantial enhancement in infectivity.Current CAR-T products primarily utilize lentiviral vector-based transduction systems to achieve CAR expression in T cells. To date, some of the safety issues observed with CAR-T therapy have been unrelated to the use of lentiviral vectors.

However, this does not imply absolute safety of the lentiviral system. While lentiviruses inherit the advantages of retroviruses, they also retain the instability associated with random genomic integration. As was the case in the late 20th century, when research advances to the stem cell level, safety concerns arising from random insertion are inevitable.

In February 2021, safety issues arose with LentiGlobin, Bluebird Bio’s product for the treatment of sickle cell disease (SCD). During the Phase 1/2 clinical trials of this product, two patients were diagnosed with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), respectively, leading to the suspension of all ongoing clinical studies involving the drug. This incident also impacted other products from Bluebird Bio. Zynteglo, its already marketed product, was also suspended from sales because it uses the same lentiviral vector as LentiGlobin.

Bluebird is not the only company to have encountered safety issues; Orchard, another rare disease drug developer, has faced nearly identical problems. In October 2020, Orchard disclosed that Strimvelis, a therapy it acquired from GSK, may have caused leukemia in one patient. Strimvelis was first approved by the European Union in 2016 for the treatment of ADA-SCID. Following this incident, sales of Strimvelis were naturally suspended.

Once again involving retroviruses (lentiviruses), hematopoietic stem cells, and hematologic malignancies, the safety concerns arising from random genomic insertion have resurfaced after two decades, potentially indicating that lentiviral vectors are not the optimal solution for the curative treatment of hereditary rare diseases.

However, from another perspective, most patients who opt for gene therapy do so because they lack suitable bone marrow matches and have no better treatment alternatives. Thus, these patients face a dilemma: should they continue to endure the suffering caused by the disease, or should they seize the opportunity for a cure despite the risk of developing hematologic malignancies?Therapeutic regimens for hematologic disorders employing lentiviral delivery technology may indeed carry unavoidable risks, but they at least offer these patients an option.

After adenoviruses brought the first wave of gene therapy to an end, this viral vector did not completely fade from the scene. In fact, through continuous optimization, the immunogenicity of adenoviruses has been gradually reduced and is now largely controllable. However, in many applications where adenoviruses were previously used, adeno-associated viruses (AAVs) have been found to offer superior efficacy and a better safety profile; consequently, AAVs are now more widely employed as vectors in gene therapy.

However, adenoviruses also have their own advantages.First, unlike lentiviral systems, genes delivered by adenoviruses do not integrate into the host genome, thereby eliminating the risk of random insertion. Adenoviral vectors also have the largest packaging capacity among various viral vector types, accommodating exogenous fragments of up to 7.5 kb. Furthermore, adenoviral vectors exhibit very high transduction efficiency and rapid post-infection expression kinetics.

Based on these characteristics, adenoviruses quickly found new applications in the field of vaccines. The research, development, and manufacturing processes for adenovirus vaccines are relatively straightforward; they enable robust expression of antigenic proteins shortly after injection and are subsequently metabolized as cells divide and replicate over time.

During the COVID-19 pandemic, CanSino Biologics, AstraZeneca, and Johnson & Johnson all opted for adenovirus vector-based COVID-19 vaccines. However, safety concerns emerged with adenovirus vectors in real-world use. Both Johnson & Johnson’s and AstraZeneca’s vaccines have repeatedly faced safety issues. Among the three products, CanSino Biologics’ vaccine demonstrated the best safety profile, with virtually no serious adverse events reported; nevertheless, its incidence of post-vaccination adverse reactions was significantly higher than that of widely used inactivated vaccines.Therefore, the value of adenoviral vector technology is beyond doubt; however, further optimization of vector safety may still be required in the future.

With the successive approvals of several gene therapy products, adeno-associated virus (AAV) as a novel viral vector delivery technology has been repeatedly highlighted. The safety and clinical value of AAV have also been demonstrated in the clinical use of these products.

Compared with adenoviruses, AAV addresses the most significant issue associated with adenoviruses—immunogenicity. AAV exhibits low immunogenicity and negligible pathogenicity in humans. This implies that the use of AAV vectors does not elicit severe immune responses, and their retained viral structures are non-pathogenic, thereby substantially enhancing the safety profile of AAV vectors.

Moreover, the currently used AAV vectors have lost the ability of wild-type AAV to integrate into the genome; the delivered transgene persists in the nucleus as head-to-tail circular DNA, further enhancing the safety profile of AAV vectors.

Due to differences in capsid proteins, AAV vectors of different serotypes exhibit varying transduction efficiencies across different tissues and cells. By selecting the appropriate AAV vector serotype, precise delivery to specific tissues and organs can be achieved.

Certainly, the drawbacks of AAV vectors are also quite apparent: first, their cargo capacity is relatively small; second, the time from infection to expression is relatively long. However, these limitations have not yet hindered the industrial application of AAV vectors.

Several drugs utilizing adeno-associated virus (AAV) vectors have been approved in succession. Glybera, which received marketing authorization from the European Union in 2012, was the first approved AAV-based therapy. Subsequently, in 2017, the U.S. Food and Drug Administration (FDA) approved Spark Therapeutics’ Luxturna for marketing. In 2019, the U.S. FDA further approved Novartis subsidiary AveXis’s Zolgensma for marketing.

If we were to identify the commonalities among these three products, apart from utilizing the same delivery system, another defining characteristic is their exorbitant cost. The prices of all three drugs are astronomical; Zolgensma is priced at a staggering $2.125 million, making it the most expensive drug to date. Glybera, due to its prohibitive pricing, saw no uptake and was ultimately withdrawn from the market in obscurity.

Product pricing must take into account multiple factors, including production costs, promotional expenses, sunk R&D costs, and the size of the patient population for the indicated indications. We do not intend to delve extensively into product pricing issues here; however, one point is clear:The production cost of AAV vectors is not a key factor contributing to the exorbitant final prices of these products.. Moreover, based on the performance of several products to date, AAV appears to be highly suited for this application scenario.

Although the aforementioned three viral vectors have not yet been widely used in clinical practice in China, numerous gene and cell therapy companies have already made relevant strategic deployments.In fact, apart from a small number of companies that achieve delivery via electroporation, the vast majority of gene and cell therapy companies must confront the challenges of viral vector development.

As discussed in the previous section, adenoviral vectors are currently primarily used in the vaccine field; lentiviral vectors are mainly applied in cell therapy; and AAV vectors are predominantly utilized in gene therapy. Setting aside adenoviral vectors, which have relatively limited applications, lentiviral and AAV vectors exhibit significant differences in industrial maturity within China, owing to the distinct developmental stages of their respective application scenarios.

In the past two years, driven by the surge in cell therapy, the industrialization of lentiviruses has advanced rapidly. The research and development, design, and manufacturing of lentiviruses have established a well-defined standard operating procedure (SOP) system, which has extended outward and facilitated the rapid emergence of the contract development and manufacturing organization (CDMO) sector for cell therapies.

On the other hand, the AAV R&D outsourcing industry has begun to emerge; however, due to its relatively early stage of development, there is no clear consensus within the sector. Optimizing GMP manufacturing of AAV vectors remains a key focus for many gene therapy companies.

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