Peer review of: Structural characterization of a novel human adeno-associated virus capsid with neurotropic prop (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
Remarks to author:


I have read this manuscript with great interest. Effective passage of blood-brain 
barrier (BBB) and establishment of infection in brain and neurological system are 
critical for the AAA therapeutic usage to treat neurological diseases. In this 
manuscript, Gao and colleagues identified a novel capsid AAVv66 from human brain 
sample by third-generation sequencing. Although AAVv66 shares a 98% sequence 
similarity with AAV2, it can much more effectively setup infection in brain in animal 
model. The study made a thorough comparison between novel AAVv66 and AAV2 in 
viral production, tissue tropism, serologic behavior and viral particle physical 
property with both in-vitro and in-vivo assays. And the analysis on structural 
information of AAVv66 further elucidate the molecular mechanism of major 
determinant amino acids contributing to the virus stability. The broad CNS 
distribution and better packaging performance of AAVv66 suggest its potential in 
CNS gene therapy. Also, the detailed structural analysis on AAVv66 can further 
leverage capsid engineering. 
 
Concerns are outlined below: 
First, in supplementary figure 5, I notice that the intravenous injection of AAVv66 
seems to have a BBB penetrating property. Are there any data that can 
demonstrate the brain tissue AAV burden (e.g. qPCR) to support AAVv66 BBB 
penetration? If so, the differences between the BBB penetrating serotype AAV9 and 
AAV PHP.B / AAV PHP.eB are worthy to be addressed, or at least have a detailed 
discussion. 
 
Second, could the transduction of AAVv66 and AAV2 be inhibited by pre-
immunization with AAVv66? This evidence should be a necessary supportive data. 
 
Third, I found it interesting that the amino acids which have most impact on viral 
particle stability or packaging efficiency are also amino acids interacting with 
various cellular receptor (e.g. E499 in AAV2 interacts with AAVR PKD2 domain; 
R585 and R588 in AAV2 interact with HSPG). However, these residues are not 
significantly involved in the inter-capsomer interaction to stabilize the virus particle. 
Could you raise a hypothesis that can possibly explain this phenomenon? 
 
Forth (related to the third point), since a set of residues which distinguish AAVv66 
and AAV2 are close to or overlapped with the residues that participate in the 
interaction with receptors (AAVR or HSPG), could it be possible that these residues 
affect receptor recognition, in addition to the impact on virus package? Moreover, a 
very interesting thing is that the AAV variants derived from AAV9 pass BBB in an 
AAVR-independent manner, but is associated with ly6a protein. I am very 
interested to know whether AAVv66 has AAVR-dependent passage of BBB, or not. 
And I am interested to know whether AAVv66 is AAVR-dependent to infect the 
normal cells, or not. Is there any difference with AAV9/PHP.eb and AAV2? Though I 
have some many questions on this point, I fully understand that this may require 
many experiments to be demonstrated. Since AAVv66 should rise great interests to 
AAV field with urgency, I suggest it could be possible that the authors give more 
discussion on this point, if they feel they could not provide these experiments in a 
short time. 
 
Minor comments: 
The punctuation marks should be used correctly. For example, the 3rd line of the 
last paragraph in Discussion “…Interestingly. AAVv66 and AAV-TT…” a comma 
should follow “interestingly” not a period. 
 
The 3rd line of section “The AAVv66 capsid is more thermostable than AAV2 across 
a range of pHs”, “uncoat” should be “uncoating”. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
The manuscript by Gao and colleagues centers on the discovery and 
characterization of a natural AAV isolate from surgically excised human tissue. The 
new strain AAVv66 shows structural and functional properties that highlight three 
critical findings - (1) surgically excised human tissue is an invaluable resource for 
discovery of new AAV strains with clinical potential in gene therapy applications; (2) 
heparin binding properties and electrostatics affect viral spread within the CNS; and 
(3) provide a roadmap for structural manipulation of other AAV strains that might 
improve their functional properties. The new strain v66 should be of immediate 
interest to the CNS gene therapy community for application in epilepsy and other 
similar focal brain indications. The study is rigorous and technically robust - a 
comprehensive analysis of a new AAV strain ranging from discovery, biophysical 
and structural analysis as well as functional characterization in mice. Some 
subjective concerns are noted below. There are a number of new AAV strains (e.g., 
AAV-TT, AAV-retro, other AAV2 heparin binding mutants in general) currently under 
evaluation. It is somewhat well known in the virology field that lower affinity to viral 
receptors enables increased spread and virulence. Additional studies in non-human 
primates might be essential to determine whether these properties could translate 
into the clinic. Otherwise, a thorough and technically rigorous study of a new AAV 
isolate.