Adeno-Associated Viral Vectors (AAV) and Their Applications in Lung Diseases

Adeno-associated virus (AAV) belongs to the family of parvoviridae, the viral particles have no envelope, and the structure is an ortho icosahedron with a diameter of about 20 nm. It is the simplest class of linear single-stranded DNA viruses discovered so far and is unable to replicate autonomously, which is infectious to both dividing and non-dividing cells. The targeted mutation of AAV capsid protein Cap gene by artificial evolutionary approach can screen AAV vectors with high transduction efficiency and different infection characteristics, which greatly increases the potential of AAV application. The recombination adeno-associated virus (rAAV) vectors used in the study are gene expression vectors modified on the basis of non-pathogenic wild-type AAV, which are characterized by variety, low immunogenicity, high safety, wide host range, high spreading ability, and long-time of exogenous gene expression in vivo, and have been widely used in gene expression, gene manipulation and gene therapy at animal level.

Studies have shown that AAV5, AAV6 and AAV9 have a high tropism for the lung. Currently, AAV has been used in clinical trials of pulmonary cystic fibrosis. The DNA contained in rAAV is generally replaced with exogenous gene expression elements for the coding genes of the AAV, retaining only the ITR sequences required for viral replication and packaging. The rAAV carrying the exogenous DNA is generated by packaging with a trans-compensated Rep gene, a Cap gene, and a coviral functional factor. rAAV has a vector capacity of 4.7 kb, and the size of the inserted exogenous target fragment does not exceed 2.8 kb.

Injection Method

Intranasal drip

Intranasal drip is a direct method of AAV infection that is simple and low equipment requirements and is novice friendly. However, the disadvantage is that AAV particles tend to be deposited in the upper respiratory tract (nasal passages, sinuses, pharynx) and gastrointestinal tract, and if effective infection of the lower respiratory tract is required, a larger dose of AAV may be needed to achieve this.

Endoscopic-assisted oral tracheal intubation

Endoscopic tracheal intubation enables AAV to be instilled directly into the lungs without surgical risk and avoids loss of viral particles from the upper airways. Since almost all AAV viral particles reach the lungs, the amount of AAV used is strictly controlled. Also, oral tracheal intubation requires some special equipment and is slightly more difficult to perform than intranasal drip.

Endotracheal injection

Compared with intranasal drip, endotracheal injection improves the efficiency of AAV entry into the lungs; and the equipment is simpler and easier to perform than the oral tracheal intubation method. However, endotracheal injection carries very high surgical risks, including bleeding, incision dehiscence, infection, and a longer recovery period. Not only that, due to the narrow trachea of mice, it is also easy to make mistakes and inject AAV into the peritracheal tissue or esophagus.

Application Cases

Case 1

Species	Method	AAV serotype	Dose	Promoter
Mice	nasal inhalation	AAV6	5x10*10vg	CAG

8-10-week-old C57BL/6 mice were introduced into the AAV6-CAG-AP vector by nasal inhalation, and the AP staining of lung tissues taken 1 month later showed that nasal epithelial cells, bronchial airway epithelial cells, and distal airway epithelial cells were efficiently transduced, and almost all alveolar cells in most regions of the lungs were stained as well, resulting in potent transduction from the nasal epithelium to the distal alveoli.

Adeno-Associated Viral Vectors (AAV) and Their Applications in Lung Diseases

Case 2

Species	Method	AAV serotype	Dose	Promoter
Mice	nasal inhalation	AAV6	5x10*10vg, 50μl	CAG

Anti-inflammatory and antifibrotic effects of IL10 on bleomycin-induced pulmonary fibrosis

Male C57BL/6J mice aged 10-12 weeks and weighing 25-30 g were injected with AAV6-CAG-IL10 or buffer control intraductally on day 0, and subcutaneously implanted with osmotic minipumps to administer 125 mg/kg bleomycin (BLM) or saline for one week consecutively on day 7, and were randomly divided into four groups, namely, Group1 (no carrier + saline), Group2 ( IL10 overexpression + saline), Group3 (no carrier + BLM) and Group4 (IL10 overexpression + BLM), and were analyzed on day 35 (3 weeks after the end of BLM treatment). H&E staining of left lung tissue sections indicated that moderate to severe inflammatory infiltration around fine bronchioles and blood vessels was observed in Group3, compared with reduced inflammatory cell infiltration in Group4, and no significant inflammatory response was observed in either Group1 or Group2.Masson trichrome staining showed that Group3 mice with subpleural areas of alveolar structural disruption and interstitial fibrosis with significant fibrotic changes, the same Group4 mice showed reduced fibrotic changes and little fibrotic manifestations were observed in the lung tissue of Group1 and Group2 mice. Quantification by the Ashcroft fibrosis score revealed that pretreatment with AAV6-CAG-IL10 significantly reduced the score, i.e., indicated improvement of BLM-induced fibrotic injury in the lungs.

To assess the effect of AAV6-CAG-IL10 vector on bleomycin-induced inflammatory response, mouse BALF (bronchoalveolar lavage fluid) was obtained on day 35 and subjected to cellular analysis, which showed that the number of inflammatory cells, such as macrophages, lymphocytes, and neutrophils, was significantly higher in Group3 and Group4 than in Group1 and Group2, while AAV6-CAG -IL10 pretreatment did not affect inflammatory cells in BALF. Total cell counts and cell sorting numbers in BALF were similar in bleomycin-treated mice with or without IL10 gene delivery.

Adeno-Associated Viral Vectors (AAV) and Their Applications in Lung Diseases

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AAV00023Z	LacZ Adeno-associated virus (AAV Serotype 1)	Inquiry
AAV00025Z	GFP Adeno-associated virus (AAV Serotype 2)	Inquiry
AAV00026Z	LacZ Adeno-associated virus (AAV Serotype 2)	Inquiry
AAV00029Z	GFP Adeno-associated virus (AAV Serotype 5)	Inquiry
AAV00030Z	LacZ Adeno-associated virus (AAV Serotype 5)	Inquiry
AAV00033Z	GFP Adeno-associated virus (AAV Serotype 6)	Inquiry
AAV00034Z	LacZ Adeno-associated virus (AAV Serotype 6)	Inquiry
AAV00035Z	LacZ Adeno-associated virus (AAV Serotype 8)	Inquiry
AAV00038Z	GFP Adeno-associated virus (AAV Serotype 8)	Inquiry

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Reference:

Kurosaki, F.; et al. AAV6-mediated IL-10 expression in the lung ameliorates bleomycin-induced pulmonary fibrosis in mice. Human gene therapy. 2018, 29(11): 1242-1251.

* For research use only. Not intended for any clinical use.

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Adeno-Associated Viral Vectors (AAV) and Their Applications in Lung Diseases

Injection Method

Application Cases

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