Anis H. Khimani, PhD
Anis H. Khimani, PhD
Senior Strategy Leader, PerkinElmer Life Sciences

Gene delivery vehicles have helped realize the concept of treating human diseases by introducing normal alleles of genes into appropriate target cells. These gene delivery vehicles include recombinant and nonrecombinant lentiviral vectors and adeno-associated virus (AAV) vectors.

Recombinant retroviral vectors have been used in clinical trials for nearly three decades. Initial results with retroviral vectors were encouraging,1,2 but the use of these vectors in nonhuman primate studies was reported to lead to T-cell lymphoma.3 These vectors were also implicated in the development of T-cell leukemia in several children who received gene therapy for X-linked severe combined immunodeficiency in clinical trials.4,5

Christian Thirion, PhD
Christian Thirion, PhD
CEO, Sirion Biotech

Adenoviral vectors in gene therapy of cystic fibrosis were reported to lack efficacy.6 Also, an adenoviral vector was suspected of playing a role in the death of a patient in a trial for gene therapy of ornithine transcarbamylase deficiency.7

Additionally, early-generation lentiviral vectors have shown clinical efficacy in more than 300 patients who received gene therapy for a number of diseases8–10; however, a recent gene therapy trial to treat cerebral adrenoleukodystrophy was halted after a participant in the study developed myelodysplastic syndrome, a bone marrow disorder that can lead to leukemia.11

Arun Srivastava, PhD
Arun Srivastava, PhD
Professor of Genetics, University of Florida College of Medicine

Despite the good safety profile of third-generation lentiviral vectors in gene therapy trials, the insertion of vector genomes in transcriptionally active sites harbors an inherent genotoxic potential, especially in patients with preexisting acquired somatic mutations, and when ubiquitously active strong promoters are used, which can activate the expression of nearby genes. Development of optimized gene expression cassettes with high and restricted activity in differentiated or specialized cells is, therefore, an important safety feature of gene therapy vectors.

Meanwhile, recombinant AAV vectors, based on a nonpathogenic parvovirus, have been used or are currently in use in 264 Phase I/II/III clinical trials for diseases such as cystic fibrosis, Batten’s disease, α1-antitrypsin deficiency, Parkinson’s disease, Pompe’s disease, and Duchenne’s muscular dystrophy.12 In some cases, such as Leber’s congenital amaurosis (LCA),13 hemophilia B,14 lipoprotein lipase deficiency,15 aromatic L-amino acid decarboxylase deficiency,16 choroideremia,17 Leber hereditary optic neuropathy,18 hemophilia A,19 and spinal muscular atrophy (SMA),20 unexpected, remarkable clinical efficacy has also been achieved.

Thus far, two AAV “drugs”—Luxturna for LCA and Zolgensma for SMA—have been approved by the FDA.21 However, in some cases, relatively large vector doses are needed to achieve clinical efficacy. The use of high doses has been shown to provoke host immune responses culminating in serious adverse events22 including the deaths of four patients.23

Although gene therapy with AAV vectors continues to be a promising treatment modality, it has also become increasingly clear that none of the first generation of AAV vectors currently in use is ideal for the following reasons:

  1. The use of AAV vectors composed of naturally occurring capsids induces immune responses, especially at high doses, because the host immune system cannot distinguish between AAV as a virus and AAV as a vector.24
  2. Like the naturally occurring AAV, most of the recombinant vectors contain single-stranded DNA, which is known to be transcriptionally inactive. Viral second-strand DNA synthesis is known to be a rate-limiting step during AAV-vector-mediated transgene expression.25
  3. Most of the AAV serotype vectors currently in use lack selective tropism for human cells and organs.

Next-generation AAVs come into focus

These limitations have been addressed in various ways. First, scientists have developed capsid-modified next-generation (NextGen) AAV serotype vectors. (These vectors are up to 80-fold more efficacious at reduced doses26,27; they are also less immunogenic.28) Second, scientists developed genome-modified generation X (GenX) AAV vectors. (These vectors mediate up to eightfold enhanced transgene expression.29) Third, scientists have combined both strategies to produce optimized AAV serotype vectors. (These vectors are about 20–30-fold more efficient at further reduced doses.30) Fourth, scientists have identified AAV3 and AAV6, serotypes that possess remarkable tropisms for human liver and primary human hematopoietic stem cells, respectively.31–36

In vivo evolution of large AAV libraries in combination with massive parallel screening of barcoded AAV vectors are now being actively employed to identify AAV vectors with further enhanced transduction properties and improved specificity.37,38

AAV serotype considerations

Multiple AAV serotypes have been isolated from tissue culture stocks from humans and nonhuman primates.39 To date, 13 distinct AAV serotype vectors (AAV1–AAV13) have been described, and this number is likely to grow. The precise mechanism of tissue tropism of AAV serotype vectors in vivo remains unknown, but it is clear that attachment to putative cell surface receptors is the initial step for successful transduction. A wealth of information has been obtained from studies in mice, where different AAV serotype vectors have been shown to exhibit distinct tropism for various tissues and organs. The efficacy of some of the AAV serotype vectors has also been evaluated in other animals, small and large, such as rodents, canines, and nonhuman primates.40

recombinant AAV serotype vectors that have been used in various animal models
In this schematic illustration, the recombinant AAV serotype vectors that have been used in various animal models and Phase I/II/III trials in humans are indicated. The animal models shown are the ones most commonly used to evaluate vector safety and efficacy. Also indicated are the organs that were targeted by the different vectors.

Innovative tools to advance discovery

For therapeutic vector design, optimization of the size of the packaged therapeutic expression cassette can be achieved. Expression cassettes that are oversized (>5 kb) can result in the packaging of truncated genomes,41 whereas packaging of expression cassettes that are undersized can result in increased cross-packaging of plasmid-derived prokaryotic sequences that incorporate antibiotic resistance genes.42 Inclusion of large non-immunogenic inverted terminal repeat (ITR)-flanking spacer sequences significantly reduces unwanted packaging of plasmid-derived prokaryotic sequences.

The design, development, and production workflow for gene therapy vectors such as AAV is similar to the workflow for classical large-molecule biotherapeutics. Living producer systems that support an efficient, sustainable, and scalable growth environment for the vectors are desirable. Such systems can drive the development of the surrounding infrastructure for workflow management, automation, and regulatory compliance.

A commonly used cell system for AAV transient transfection and production is HEK293. For other producer biological model systems, either mammalian or insect cell lines have been used to scale up AAV sequences containing the transgene. For stable mammalian cell lines, either BHK cells or HeLa cells are used. With the insect system, Sf6 cells infected with recombinant baculovirus have been used as well to scale up recombinant AAV. With either of the above cell systems, production levels of 104 to 105 genome copies/L are obtained.

Viral vector characterization is also an important application in gene therapy. It facilitates the evaluation of critical quality attributes (CQAs) such as identity, potency, purity, and stability. To evaluate CQAs and comply with regulatory guidelines, companies engaged in gene therapy need to adopt reproducible and robust methods of development and validation.

A number of analytical techniques enable chemical and physical characterization. Viral structure and particle integrity and aggregation can be assessed with transmission electron microscopy (TEM), contributing to the quality control of viral vectors. TEM can also be used in combination with molecular techniques such as droplet digital PCR (ddPCR) or quantitative real time PCR (qPCR) to determine AAV identity and purity.

Recent attention has been focused on analytical ultracentrifugation and mass spectrometry as well. However, some of these technologies have limited throughput. Higher throughput microfluidic capillary electrophoresis platforms, such as the LabChip GXII Touch with the ProteinEXact assay, can enable AAV capsid protein (VP1, VP2, VP3) analysis.

Some of the above technologies are critical to the orthogonal validation of conventional or well-established methods such as electrophoresis, spectroscopy, and chromatography.

Artificial intelligence and machine learning, along with analytical data management solutions, are significantly supporting analytical techniques to achieve automation, predictability, and decision making along the vector development, characterization, scale up, and QA/QC workflows.

Advanced tools, methods, applications, and services can facilitate the design, manufacture, and characterization of gene therapy vectors. End-to-end solutions can incorporate platforms for liquid handling and microfluidics; platforms for the analysis of macromolecules; platforms for cellular analysis and in vivo imaging; as well as platforms for bioinformatics applications. In addition, enhanced security software tools that enable 21 CFR Part 11 compliance to meet the regulatory requirements within the GMP lot release environment are critical.

Therapeutic gene and cell therapy research and development can progress more readily when advanced technologies and services for viral vector design and manufacturing are adopted. Such technologies, in combination with technologies for CRISPR-based gene editing, RNA interference, base editing, and prime editing, can move innovative therapeutics forward.


Anis H. Khimani, PhD, is the senior strategy leader for pharmaceutical development at PerkinElmer Life Sciences. Christian Thirion, PhD, is the CEO of Sirion Biotech. Arun Srivastava, PhD, is the George H. Kitzman Professor of Genetics at the University of Florida College of Medicine.


1. Rosenberg SA, Abersold P, Cornetta K, et al. Gene transfer into humans—Immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction. N. Engl. J. Med. 1990; 323: 570.
2. Grossman M, Raper SE, Kozarsky K, et al. Successful ex vivo gene therapy directed to liver in a patient with familial hypercholesterolemia. Nat. Genet. 1994; 6: 335.
3. Donahue RE, Kessler SW, Bodine D, et al. Helper virus induced T cell lymphoma in non-human primates after retroviral mediated gene transfer. J. Exp. Med. 1992; 176: 1125.
4. Hacein-Bey-Abina S, Von Kalle C, Schmidt M, et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 2003; 302: 415–419.
5. Ott MG, Schmidt M, Schwarzwaelder K, et al. Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat. Med. 2006; 12: 401–409.
6. Knowles MR, Hohneker KW, Zhou Z, et al. A controlled study of adenoviral vector-mediated gene transfer in the nasal epithelium of patients with cystic fibrosis. N. Engl. J. Med. 1995; 333: 823.
7. Raper SE, Chirmule N, Lee FS, et al. Fatal systemic inflammatory response syndrome in an ornithine transcarbamylase deficient patient following adenoviral gene transfer. Mol. Genet. Metabol. 2003; 80: 148–158.
8. Cartier N, Hacein-Bey-Abina S, Bartholomae CC, et al. Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science 2009; 326: 818–823.
9. Cavazzana-Calvo M, Payen E, Negre O, et al. Transfusion independence and HMGA2 activation after gene therapy of human β-thalassemia. Nature 2010; 467: 318–322.
10. Ribeil JA, Hacein-Bey-Abina S, Payen E, et al. Gene therapy in a patient with sickle cell disease. N. Engl. J. Med. 2017; 376: 848–855.
11. Servick K. Gene therapy clinical trial halted as cancer risk surfaces. Science. DOI: 10.1126/science.abl8782, 2021.
12. Mendell JR, Al-Zaidy SA, Rodino-Klapac LR, et al. Current clinical applications of in vivo gene therapy with AAVs. Mol. Ther. 2021; 29: 464–488.
13. Bainbridge JW, Smith AJ, Barker SS, et al. Effect of gene therapy on visual function in Leber’s congenital amaurosis. N. Engl. J. Med. 2008; 358: 2231–2239.
14. Nathwani AC, Tuddenham EG, Rangarajan S, et al. Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N. Engl. J. Med. 2011; 365: 2357–2365.
15. Gaudet D, Methot J, Dery S, et al. Efficacy and long-term safety of alipogene tiparvovec (AAV1-LPLS447x) gene therapy for lipoprotein lipase deficiency: An open-label trial. Gene Ther. 2013; 20: 361–369.
16. Hwu WL, Muramatsu S, Tseng SH, et al. Gene therapy for aromatic l-amino acid decarboxylase deficiency. Sci. Transl. Med. 2012; 4: 134ra161.
17. MacLaren RE, Groppe M, Barnard AR, et al. Retinal gene therapy in patients with choroideremia: Initial findings from a phase 1/2 clinical trial. Lancet 2014; 383: 1129–1137.
18. Feuer WJ, Schiffman JC, Davis JL, et al. Gene Therapy for Leber Hereditary Optic Neuropathy: Initial Results. Ophthalmology 2016; 123: 558–570.
19. Rangarajan S, Walsh L, Lester W, et al. AAV5-Factor VIII Gene Transfer in Severe Hemophilia A. N. Engl. J. Med. 2017; 377: 2519–2530.
20. Mendell JR, Al-Zaidy S, Shell R, et al. Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy. N. Engl. J. Med. 2017; 377: 1713–1722.
21. Keeler AM, Flotte TR. Recombinant adeno-associated virus gene therapy in light of Luxturna (and Zolgensma and Glybera): Where are we, and how did we get here? Annu. Rev. Virol. 2019; 6: 601–621.
22. Wilson JM, Flotte TR. Moving forward after two deaths in a gene therapy trial of myotubular myopathy. Hum. Gene. Ther. 2020; 31: 695–696.
23. Shieh PB, Bonnemann CG, Muller-Felber W, et al. Letter to the Editor. Hum. Gene Ther. 2020; 31: 787.
24. Srivastava A. Adeno-associated virus: The naturally occurring virus versus the recombinant vector. Hum. Gene Ther. 2016; 27: 1–6.
25. Qing K, Wang XS, Kube DM, et al. Role of tyrosine phosphorylation of a cellular protein in adeno-associated virus 2-mediated transgene expression. Proc. Natl. Acad. Sci. USA 1997; 94: 10879–10884.
26. Zhong L, Li B, Mah CS, et al. Next generation of adeno-associated virus 2 vectors: point mutations in tyrosines lead to high-efficiency transduction at lower doses. Proc. Natl. Acad. Sci. USA 2008; 105: 7827–7832.
27. Markusic DM, Herzog RW, Aslanidi GV, et al. High-efficiency transduction and correction of murine hemophilia B using AAV2 vectors devoid of multiple surface-exposed tyrosines. Mol. Ther. 2010; 18: 2048–2056.
28. Martino AT, Basner-Tschakarjan E, Markusic DM, et al. Engineered AAV vector minimizes in vivo targeting of transduced hepatocytes by capsid-specific CD8+ T cells. Blood 2013; 121: 2224–2233.
29. Ling C, Wang Y, Lu Y, et al. Enhanced transgene expression from recombinant single-stranded D-sequence-substituted adeno-associated virus vectors in human cell lines in vitro and in murine hepatocytes in vivo. J. Virol. 2015; 89: 952–961.
30. Ling C, Li B, Ma W, et al. Development of Optimized AAV serotype vectors for high-efficiency transduction at further reduced doses. Hum. Gene Ther. Methods 2016; 27: 143–149.
31. Glushakova LG, Lisankie MJ, Eruslanov EB, et al. AAV3-mediated transfer and expression of the pyruvate dehydrogenase E1 alpha subunit gene causes metabolic remodeling and apoptosis of human liver cancer cells. Mol. Genet. Metab. 2009; 98: 289–299.
32. Vercauteren K, Hoffman BE, Zolotukhin I, et al. Superior in vivo transduction of human hepatocytes using engineered AAV3 capsid. Mol. Ther. 2016; 24: 1042–1049.
33. Brown HC, Doering CB, Herzog RW, et al. Development of a clinical candidate AAV3 vector for gene therapy of hemophilia B. Hum. Gene Ther. 2020; 31: 1114–1123.
34. Kumar SRP, Xie J, Hu S, et al. Coagulation factor IX gene transfer to non-human primates using engineered AAV3 capsid and hepatic optimized expression cassette. Mol. Ther. Methods Clin. Devel. 2021; 23: 98–107.
35. Song L, Li X, Jayandharan GR, et al. High-efficiency transduction of primary human hematopoietic stem cells and erythroid lineage-restricted expression by optimized AAV6 serotype vectors in vitro and in a murine xenograft model in vivo. PLoS One 2013; 8: e58757.
36. Yang H, Qing K, Keeler GD, et al. Enhanced transduction of human hematopoietic stem cells by AAV6 vectors: Implications in gene therapy and genome editing. Mol. Ther. Nucl. Acids 2020; 20: 451–458.
37. Herrmann AK, Bender C, Kienle E, et al. A robust and all-inclusive pipeline for shuffling of adeno-associated viruses. ACS Synth. Biol. 2019; 8(1): 194–206. DOI: 10.1021/acssynbio.8b00373.
38. Weinmann J, Weis S, Sippel J, et al. Identification of a myotropic AAV by massively parallel in vivo evaluation of barcoded capsid variants. Nat. Commun. 2020; 11(1): 5432. DOI: 10.1038/s41467-020-19230-w.
39. Gao GP, Alvira MR, Wang L, et al. Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc. Natl. Acad. Sci. USA 2002; 99: 11854–11859.
40. Srivastava A. In vivo tissue-tropism of adeno-associated viral vectors. Curr. Opin. Virol. 2016; 21: 75–80.
41. Dong B, Nakai H, Xiao W. Characterization of genome integrity for oversized recombinant AAV vector. Mol. Ther. 2010; 18: 87–92.
42. Chadeuf G, Ciron C, Moullier P, Salvetti A. Evidence for encapsidation of prokaryotic sequences during recombinant adeno-associated virus production and their in vivo persistence after vector delivery. Mol. Ther. 2005; 12: 744–753.

Previous articleNew Cancer Treatments Lead to New Strategic Options
Next articleNovel Gene Editing Systems Come Into Their Own